# Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals

Mathias Girbig, Juanjuan Xie, Helga Grötsch, Domenico Libri, Odil Porrua, Christoph Müller

► To cite this version:

Mathias Girbig, Juanjuan Xie, Helga Grötsch, Domenico Libri, Odil Porrua, et al. Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals. Cell Reports, 2022, 40 (10), pp.111316. 10.1016/j.celrep.2022.111316. hal-03853244

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# Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals

## Graphical abstract

<figure><img src="image_1.png" /></figure>

## Authors

Mathias Girbig, Juanjuan Xie, Helga Grötsch, Domenico Libri, Odil Porrua, Christoph W. Müller

## Correspondence

christoph.mueller@embl.de

## In brief

RNA polymerase III (Pol III) terminates transcription of small RNAs on polythymine sequences on the non-template DNA strand. Girbig et al. report and functionally validate the structure of the yeast Pol III pre-termination complex, thereby revealing how termination signal recognition and transcription pausing, a critical prerequisite for termination, is achieved.

## Highlights

* We report the cryo-EM structure of the yeast RNA polymerase III pre-termination complex

* Polar interactions between subunit C128 and the poly(dT) termination site induce pausing

* Mutating key interaction residues in C128 impairs termination *in vitro* and *in vivo*

* Subcomplex C53-C37 does not bind poly(dT) but assists in its recognition through C128

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

Article

# Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals

Mathias Girbig,<fnref n="1" />,<fnref n="3" /> Juanjuan Xie,<fnref n="2" />,<fnref n="4" /> Helga Grötsch,<fnref n="1" />,<fnref n="5" /> Domenico Libri,<fnref n="2" />,<fnref n="6" /> Odil Porrua,<fnref n="2" />,<fnref n="6" /> and Christoph W. Müller<fnref n="1" />,<fnref n="7" />,*

## SUMMARY

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7–3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.

## INTRODUCTION

Eukaryotic RNA transcription is carried out by multiple DNA-dependent RNA polymerases (Pols). Pol I transcribes ribosomal RNA (rRNA). Pol II produces messenger RNAs (mRNAs) and various non-coding RNAs. Pol III synthesizes short, abundant RNA molecules such as transfer RNAs (tRNAs), the U6 small nuclear RNA (snRNA), and the 5S rRNA. Pol III is specialized to cope with the high demand of its target transcripts and, as such, requires fewer factors to initiate, elongate, and terminate transcription.

Pol III, similar to Pol I and Pol II, contains a 10-subunit core and a 2-subunit stalk domain. In addition, it features the C53-C37 heterodimer and the C82-C34-C31 heterotrimer. C53-C37 shares structural homology with the Pol II general transcription factor (GTF) TFIIF. Pol III also harbors subunit C11 (RPC10 in humans), whose C-terminal domain is homologous to the RNA-cleaving domain of the Pol II elongation factor TFIIS (Chedin et al., 1998). Similar to TFIIF and TFIIE, C53-C37 and C82-C34-C31 are required to initiate transcription (Brun et al., 1997; Kassavetis et al., 2010; Landrieux et al., 2006; Lefèvre et al., 2011; Thuillier et al., 1995; Wang and Roeder, 1997). C53-C37 and C11 are also crucial for Pol III transcription termination (Arim-

basseri and Maraia, 2013, 2015; Chedin et al., 1998; Landrieux et al., 2006; Mishra and Maraia, 2019; Mishra et al., 2021; Rijal and Maraia, 2013).

Transcription termination is the final step in the transcription cycle and involves the release of DNA and RNA. As for Pol I and Pol II, a multitude of accessory factors are needed for transcription termination (Porrua and Libri, 2015; Richard and Manley, 2009). In contrast, Pol III relies on fewer elements to terminate transcription. Except for the helicase Sen1, which was recently reported to function in Pol III transcription termination (Rivosecchi et al., 2019; Xie et al., 2022) and likely acts as a “fail-safe” factor to help the release of Pol III molecules that fail to terminate in the first place (Xie et al., 2022), Pol III relies on only its built-in subunits and a termination sequence on the DNA to terminate transcription. The termination process allows fast recycling of Pol III to a new round of transcription in a process termed facilitated recycling (Dieci and Sentenac, 1996). Pol III terminates transcription on continuous stretches of thymine bases (dT) on the non-template (NT) strand of the DNA (Allison and Hall, 1985; Bogenhagen and Brown, 1981; Cozzarelli et al., 1983; Watson et al., 1984). Yeast Pol III requires at least 5 dTs to correctly terminate transcription (Allison and Hall, 1985) and, in vitro, does not rely on external factors to do so (Braglia

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany</footnote>

<footnote n="2">²Université de Paris, CNRS, Institut Jacques Monod, 75006 Paris, France</footnote>

<footnote n="3">³Present address: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Straße 10, 35043 Marburg, Germany</footnote>

<footnote n="4">⁴Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA</footnote>

<footnote n="5">⁵Present address: VERAXA Biotech GmbH, Carl-Friedrich-Gauß-Ring 5, 69124 Heidelberg, Germany</footnote>

<footnote n="6">⁶Present address: Institut de Génétique Moléculaire de Montpellier, Université de Montpellier, CNRS, Montpellier, France</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: christoph.mueller@embl.de
https://doi.org/10.1016/j.celrep.2022.111316</footnote>
<figure><img src="image_5.png" /></figure>

et al., 2005; Landrieux et al., 2006). Pol III uses its subunits C160 (Rijal and Maraia, 2016), C128 (James and Hall, 1990; Shaaban et al., 1995, 1996), C53-C37 (Arimbasseri and Maraia, 2013; Landrieux et al., 2006; Rijal and Maraia, 2013), and C11 (Chedin et al., 1998; Iben et al., 2011; Landrieux et al., 2006) to terminate transcription. C11 is also critical for the facilitated recycling process (Landrieux et al., 2006; Mishra et al., 2021). Prior to termination, Pol III slows down (Rijal and Maraia, 2016), to which C53-C37 contributes (Arimbasseri and Maraia, 2013). When Pol III encounters the poly(dT) termination sequence, it pauses on the 4th dT, thus forming the pre-termination complex (PTC), and C53-C37 was shown to be critical for PTC formation (Arimbasseri and Maraia, 2015). How Pol III recognizes the poly(dT) termination signal in the context of the Pol III PTC and how this facilitates pausing of Pol III and predisposes it for the termination process was, however, not known.

Structural insights into Pol III transcription could be obtained from initiating (Abascal-Palacios et al., 2018; Han et al., 2018; Vorländer et al., 2018) and elongating yeast Pol III (Hoffmann et al., 2015) via cryogenic electron microscopy (cryo-EM). In addition, structures of human Pol III were recently determined (Girbig et al., 2021; Li et al., 2021; Ramsay et al., 2020; Wang et al., 2021). A recent cryo-EM structure of the human Pol III PTC at 3.6 Å resolution showed that subunit RPC2 (the human ortholog to C128) recognizes the poly(dT) termination sequence (Hou et al., 2021). However, to what extent the contact sites between RPC2 and the termination sequence are critical to termination is still not known, due to a lack of functional validation of this interaction. Furthermore, the human Pol III PTC structure could not explain why C53-C37 (RPC4-RPC5 in humans) is critical for correct termination.

Here, we report the cryo-EM structure of the yeast Pol III PTC at a resolution of 2.8 Å. The structure confirms that C128 recognizes the poly(dT) termination signal and explains how pausing is achieved, which we extensively validate with structure-function studies. We also present complementary structures that shed light on how C53-C37 participates in transcription termination and how PTC formation may drive the subsequent termination process.

## RESULTS

### Purified yeast Pol III terminates transcription in vitro

To study the molecular mechanism of Pol III transcription termination on poly(dT) sequences, we first tested if the yeast Pol III sample used by us in earlier structural studies (Hoffmann et al., 2015; Vorländer et al., 2018, 2020) is also capable of terminating transcription on poly(dT) sequences. We used a double-stranded (ds) DNA construct composed of the S. cerevisiae U6 snDNA gene promoter and followed by the 3' flanking sequence of the S. cerevisiae tRNA gene tL(CAA)L, which contains a strong 5-dT termination signal (Braglia et al., 2005) (Figure S1A). Recruitment of Pol III to the promoter DNA was facilitated by the addition of the Pol III GTF TFIIIB. A time course showed the appearance of a strong RNA band (visualized by supplementation with radioactively labeled UTP) at the expected size of ca. 35 nt, indicating that Pol III pauses and presumably terminates on the termination site (Figure S1B). Furthermore, Pol III paused

or terminated on the 5-dT signal but not on the sequences carrying only 3 or 4 dTs (Figure S1C). Instead, termination read-through (RT) products could be observed that corresponded to transcripts that were released once Pol III reached the end of the dsDNA. While this assay does not allow us to distinguish between pausing and termination, a similarly prepared batch of purified Pol III was recently shown to efficiently terminate on a 5-dT termination sequence on immobilized DNA templates (Xie et al., 2022). Together, these results confirm that Pol III is able to pause, and presumably terminate, on its own in vitro in a promoter-dependent transcription setup.

### A 7-dT signal traps Pol III in a pre-termination state

To determine the structure of the Pol III PTC, we trapped Pol III in the PTC conformation by incubating it with a transcription scaffold that contained a 7-dT termination signal on the NT-DNA strand (Figure 1A). The scaffold also featured a 12-nt mismatch between the template (T) and the NT strand, thereby resembling an open transcription bubble. The DNA:RNA hybrid was also substituted so that it did not carry a poly(dA:rU) sequence to prevent spontaneous termination. We compared the capacity of Pol III to extend the RNA oligonucleotide in a 7-dT (PTC-like) or 2-dT (elongation complex [EC]-like) scaffold (Figure 1B). Addition of nucleoside triphosphates (NTPs) induced extension of the RNA primer on both scaffolds. However, on the EC scaffold, Pol III primarily transcribed long RNA products (lane 4), whereas a strong RNA band, just 3 bases downstream of the 3' end of the RNA primer used was detected on the PTC scaffold (lane 8). The appearance of this RNA band, which could hardly be observed on the EC scaffold, indicated that Pol III pauses in the presence of the 7-dT signal on the NT strand and that the 7-dT scaffold traps Pol III in a PTC-like state.

### Structure of the yeast Pol III PTC

Next, we assembled the yeast Pol III complex on the 7-dT PTC scaffold and acquired a high-resolution cryo-EM dataset (Data S1). Processing the data yielded a 3D reconstruction of the Pol III PTC with a nominal resolution of 2.8 Å that locally extended to 2.5 Å (Data S1). The cryo-EM map (map A in Data S1) showed a clear signal for the bound nucleic acids, with the downstream dsDNA and the DNA:RNA hybrid being well resolved (Figures 1C and S2A). The C160 bridge helix and trigger loop, which are both important active-site elements, are fully folded (Figure S2A). The C-terminal domain of C11, which was not resolved in the yeast Pol III EC structure, adopts an “outside funnel” conformation that is similar to that in apo yeast Pol III (Hoffmann et al., 2015) but differs from the “outside funnel” conformation in human Pol III (Girbig et al., 2021) (Figure S2B). Thanks to the improved quality of the 3D reconstruction of the Pol III PTC, we could extend the Pol III structural model compared with existing models. Additional densities near the heterodimer and in the heterotrimer could be assigned to C53 (aa 195–224) and C31 (aa 101–161), respectively (Figures S2C and S2D). We further assigned additional densities, one in the DNA binding cleft formed by subunit C160 and one near subunit AC40 (aa 311–329), to molecules of the detergent CHAPSO, which was used to optimize cryo-EM grid preparation (Figures S2E and S2F).
<figure><img src="image_6.png" /></figure>

Importantly, the cryo-EM reconstruction showed a strong signal of the unpaired NT strand and allowed building of the complete 7-dT termination signal (Figure 1D). Because the cryo-EM sample preparation conditions differed between the Pol III PTC and the earlier reported Pol III EC (Hoffmann et al., 2015), we determined a Pol III EC structure under exactly the same conditions as for the Pol III PTC and only reduced the number of the dTs on the NT strand. Cryo-EM data collection and processing yielded a 3D reconstruction of the Pol III EC control (map B) with a nominal resolution of 3.4 Å that locally reached 2.9 Å (Data S2). While the cryo-EM signals of unrelated polypeptide elements were of comparable qualities, the NT strand was only poorly resolved in the Pol III EC (Figure 1D). We further compared our cryo-EM reconstruction with reported structures of other eukaryotic RNA Pols. The comparison revealed that the NT strand in the Pol III PTC is also much better resolved than the NT strand in the yeast Pol I EC (Tafur et al., 2016) and yeast Pol II EC

<caption>Figure 1. Yeast Pol III PTC structure</caption>

(A) Schematic of the 7-dT transcription scaffold. Red, RNA; dark blue, T strand; light blue, NT strand.

(B) RNA primer extension experiment. The elon-
gation complex (EC) scaffold contains 2 dTs on the
NT strand (left), and the PTC scaffold contains 7
dTs (right). Image contrast was adjusted for clarity.

(C) Cryo-EM reconstruction of the yeast Pol III PTC in two different views. Nucleic acids are colored as in (A).

(D) Close-up view onto the NT-strand binding region of Pol III PTC (this study), Pol III EC (this study), Pol I EC (EMDB: 0240; PDB: 6HLR), and Pol II EC (PDB: 5C4J; 2Fobs – Fcalc calculated at 1.2 σ).

(E) Atomic model of the yeast Pol III PTC colored by root-mean-square deviation (RMSD) between the Pol III EC (not shown) and the Pol III PTC.

(F) Local contraction of subunit C128 around the NT strand upon PTC formation. Conformational changes from EC to PTC in subunit C128 are depicted as red arrows.

structures (Barnes et al., 2015) (Fig-
ure 1D). Hence, the strong signal of
the NT strand is a specific feature of
the Pol III PTC and indicates that Pol
III is capable of recognizing the poly(dT)
termination signal in a specific manner.

Superimposition with the Pol III EC
shed light on conformational changes
upon recognition of the termination
signal as shown by a local contraction
of subunit C128 around the NT strand
(Figures 1E and 1F). The contracting re-
gions of C128 can be mapped to the
C128 lobe (aa 196–370), protrusion (aa
60–195 and 371–439), and fork (aa 440–
521) domains. In addition, the C53-C37
heterodimer, which binds the C128 pro-
trusion domain, subunit C11, and the
jaw domain of C160 contract toward

the direction where the NT strand resides. Compared with the
yeast Pol I and Pol II EC structures, the conformations of the
Pol III fork (see below) and lobe domains differ considerably
in size and conformation (Figures S3A and S3B). The Pol I
lobe would clash with the modeled NT strand of the superim-
posed Pol III PTC (Figure S3A), indicating that the NT strand fol-
lows a different path in Pol I. The Rpb2 lobe region near the NT
strand was not modeled in the only yeast Pol II EC structure that
features a fully built transcription bubble (Barnes et al., 2015),
presumably because it was too flexible (Figure S3B). Conse-
quently, the path of the NT strand and its exit regions differ be-
tween Pol I, Pol II, and Pol III (Figure S3C). The NT strand in the
Pol III PTC runs along a narrow cavity, which does not form in
Pol I and Pol II. These structural differences suggest that the
NT strand is more tightly bound in Pol III (when a poly[dT] termi-
nation signal is present) than it is in Pol I and Pol II, which would
rationalize why only Pol III canonically terminates on poly(dT)
signals.
A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

D

<figure><img src="image_10.png" /><figcaption>Figure 3. Mutating Pol III C128 on specific sites interferes with Pol III termination in vitro</figcaption></figure>

(A) Protein gels of purified S. cerevisiae Pol III WT and mutant samples. Asterisks mark the tagged subunits in the C128-TAP (used for the experiments) and AC40-TAP (loaded as control) Pol III samples. Individual protein lanes are cropped from protein gels prepared after each purification.

(B) Schematic of the tailed-template DNA construct. The positions of the poly(dT) termination signal and of the first guanine base (dG) are highlighted.

(C) *In vitro* transcription experiment on tailed-template DNA constructs. Experiments on WT and mutant Pol III variants were done on either a 6-dT or a 3-dT construct. The denaturing RNA gel corresponds to one of three independent experiments. RNA bands used to quantify the Pol III transcription products are indicated by black boxes for the Pol III WT lanes. T, termination; RT, readthrough. Gel boundaries (top, bottom) were cropped and image contrast was adjusted for clarity.

(D) Quantification of the Pol III transcription products indicated in (C). Bar plots represent normalized RNA band intensities (mean ± SD) from three technical replicates. For lane 15 (K448A), two technical replicates were used.

as its substitution showed a less severe pausing/termination deficiency.

## Pol III C128 mutations affect yeast viability and cause global termination defects in vivo

We next asked whether the C128 mutations also affect Pol III function in vivo and first compared the growth of the WT and the different C128 mutant strains (Figure 4A). For the triple mutation, we observed a severe thermosensitive growth phenotype at 37°C, whereas growth was unaffected at 30°C or 25°C. We could also observe a slight thermosensitive phenotype for the R481G mutant. The Q199R displayed a clear growth defect at 30°C, which was even more pronounced at 25°C. These results support the idea that these mutations compromise Pol III function in vivo. For the K448A and R451V mutants, we did not observe any growth phenotype, suggesting that the effect of these mutations on Pol III activity is not severe enough to impair yeast growth.

We then directly assessed whether the most disruptive C128 mutations provoke Pol III termination defects in vivo. To this end, we generated high-resolution genome-wide maps of transcribing Pol III by UV cross-linking and analysis of cDNAs (CRAC), which allows mapping the position of Pol III with nucleotide resolution (Candelli et al., 2018; Granneman et al., 2009). We performed these experiments in strains carrying the WT, Q199R, R451V, or triple mutant version of C128 (Figures 4 and S4) grown at permissive temperature (i.e., 30°C). We obtained very specific and reproducible Pol III signals in the two biological replicates, with most of the reads mapping to Pol III-transcribed genes (i.e., tRNA, 5S RNA, and a few additional genes; Figure S4). Metagene analysis of Pol III distribution around tRNA

genes (Figure 4B) indicates that a considerable fraction of WT Pol IIIs read through the primary terminator (i.e., the first T track after the 3' end of the gene), in agreement with previous studies (Turowski et al., 2016; Xie et al., 2022). Importantly, we observed a significant increase in Pol III signal downstream of the primary terminator in the three mutants, indicating global transcription termination defects (Figure 4B and individual examples in Figure 4C). Clear termination defects were also observed at other Pol III-dependent genes such as the 5S and the SNR52 genes (Figure S4E). Heatmap analysis of Pol III occupancy (log2 ratio) in each mutant relative to the WT showed that such an increase in the Pol III RT signal can be detected for the vast majority of tRNA genes (Figure 4D), further confirming the generality of termination defects. Among these mutants, Q199R exhibited the most pronounced termination defects. While the R451V mutant did not show a growth phenotype, it was defective in Pol III transcription termination, similar to the triple mutant or even to a slightly higher degree for some genes. The extent of transcription termination defects of the R451V and the triple mutant might not be high enough to induce growth defects at 30°C. Yet, at 37°C, growth might be more affected in the triple mutant because its residues form (when not mutated) several H bonds with the NT-strand bases, whereas R451 does not. Hence, altering the different modes of NT-strand recognition appears to affect yeast viability in different ways, suggesting that the formation of a tight H-bond network is the prime mode of NT-strand recognition.

To perform a more quantitative assessment of termination defects caused by the different C128 mutations, we computed for each tRNA gene the RT index (determined by the ratio of Pol III signals within 500 bp downstream of the primary terminator
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

(legend on next page)
relative to the gene body, Figure 4G) in the WT and mutant strains. We observed an increase in the average RT index, and thus a decrease in the Pol III termination efficiency, in all mutants compared with the WT, with the largest increase in Q199R and a more moderate and similar increase in R451V and the triple mutant (Figure 4H). We considered the possibility that the different C128 mutations could have different impacts on the individual tRNA genes depending on the length of their terminator sequence. For instance, the Q199R mutation, predicted to prevent the interaction with the fourth T of the terminator, might not strongly affect termination at very long T tracks. To address this possibility, we analyzed the RT index on tRNA genes grouped according to the length of their primary terminator. However, we observed a similar behavior in the three mutants, namely a general increase in the RT index at all gene sets that tended to decrease as the length of the primary terminator increased (Figure 4I). Therefore, the introduced mutations reduce the efficiency of termination independent of the T-track length.

Altogether, these results indicate that mutations in C128 designed to interfere with the recognition of or the response to the poly(dT) termination signal impair Pol III transcription termination in vivo.

## Structure of Pol III Δ sheds light on the role of C53-C37 in Pol III transcription termination

The structure of the Pol III PTC revealed a key role for C128 in Pol III transcription termination. However, it did not explain the function of C53-C37, which has been demonstrated to be essential for Pol III transcription termination but does not contact the NT strand directly in the Pol III PTC. To better understand the role of C53-C37, we determined the structure of a Pol III variant lacking C53-C37 and C11 (hereafter referred to as Pol III Δ). We purified Pol III Δ from an *S. cerevisiae* strain in which the C11 gene was substituted with its *S. pombe* ortholog, thereby causing loss of C53-C37 and C11 during protein purification (Kassavetis et al., 2010) (Figure 5A). We assembled Pol III Δ on the 7-dT PTC scaffold, subjected it to cryo-EM analysis, and obtained a 3.9 Å cryo-EM map (Data S3). Despite the lower resolution, the cryo-EM map was still of sufficient quality to build the Pol III Δ structure for comparison with the Pol III PTC. The Pol III Δ structure lacked the density for C53-C37 and C11 but clearly featured cryo-EM density for the bound DNA and RNA elements (Figure 5B). The

cryo-EM signal corresponding to the NT strand was, however, much poorer than in the Pol III PTC, despite the presence of the 7-dT termination signal (Figure 5B). We further noticed that the fork, protrusion, and lobe elements of C128 adopted a relaxed, instead of a contracted, conformation (Figures 5B and 5C). Hence, the earlier described contraction of the Pol III core upon NT-strand recognition was essentially reversed in the absence of C53-C37 and C11. Instead, the conformation of the Pol III Δ core resembled that of the Pol III EC despite the presence of the termination signal (Figure 5D). We concluded from this observation that the C53-C37 heterodimer—although it does not directly contact the NT strand—indirectly assists Pol III termination by binding the lobe of C128 and positioning it for interaction with the NT strand.

## NTP addition leads to a register offset between the unwound DNA strands

Finally, we set out to determine the structure of the Pol III PTC in the presence of NTPs. We incubated Pol III with the 7-dT PTC scaffold and with an NTP mix containing CTP, ATP, and UTP and obtained a 3D reconstruction (map D) of the Pol III PTC + NTPs (Figure 6A) at a nominal resolution of 2.7 Å (Data S4). We noticed that the upstream DNA was slightly better resolved, which encouraged us to perform masked 3D classification on the nucleic acid elements (Data S4). We, thereby, obtained a 3D map (map E) with a significantly improved signal of the upstream DNA moiety that allows the visualization of the complete transcription bubble (Figure 6B).

The cryo-EM densities in the Pol III PTC and PTC + NTPs structures were of sufficient quality to define the registers of the DNA:RNA hybrids (Figures 6C and 6D). In the Pol III PTC, the 3'-end guanine (G19) of the RNA oligonucleotide used sits on position i - 1, whereas the nucleotide addition side (i + 1) position is not occupied (Figure 6C). In the Pol III PTC + NTPs structure, the RNA was extended by at least 1 nt, and the bases of the hybrid were shifted by one position away from the nucleotide addition site, which was occupied by a newly attached cytosine (Figure 6D). We also observed a weak density that occupied position i + 1 and extended the RNA model with another adenosine (A21). Surprisingly though, we did not observe a register shift on the NT strand (Figures 6D and 6E), which was also very well resolved in the Pol III PTC + NTPs structure. Hence, the addition

## Figure 4. Characterization of the effects of C128 mutations on cell growth and Pol III transcription termination in vivo

(A) Serial dilution analysis of the growth of C128 mutant strains at various temperatures. One of three technical replicates is shown. Red and blue arrows highlight heat- and cold-sensitive growth phenotypes, respectively.

(B) Metagene analysis of the Pol III distribution around tRNA genes. The signal covering the region between the 5' end and the primary terminator (i.e., the first T track after the 3' end of the mature tRNA) is scaled to 100 bp. Values on the y axis correspond to the mean coverage expressed as counts per million (CPM). Data are represented in the logarithmic scale. Two technical replicates were performed.

(C) Integrative Genomics Viewer (IGV) screenshots of examples of tRNA genes displaying termination defects in the indicated strains. Values within brackets correspond to the scale of the Pol III signal expressed in CPM. Only the signal on the strand hosting the tRNA gene is indicated.

(D-F) Heatmap analyses representing the log2 fold change (FC) of the Pol III signal in the indicated mutants relative to the WT at tRNA genes. The summary plots on the top were calculated using the average values for each position. tRNA genes in the heatmaps are sorted in the same order determined by the ranking of signals in (D).

(G) Scheme of a tRNA transcription unit indicating the gene body and the readthrough (RT) region.

(H) Comparison of the average RT index for tRNA genes in the indicated strains. “p” corresponds to the p value for the global comparison of the four groups according to the Kruskal-Wallis test. Asterisks denote the p values of pairwise comparisons ("p ≤ 0.05; ****p ≤ 0.0001").

(I) RT index of tRNA genes grouped according to the T-track length of their primary terminator in the indicated strains. Data points correspond to the average value, whereas error bars denote the standard error.
<figure><img src="image_20.png" /><figcaption>Figure 5. Cryo-EM structure of Pol III Δ</figcaption></figure>

(A) Protein gel of Pol III WT and Pol III Δ. Figure modified from Xie et al. (2022) © the authors, some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license (http://creativecommons.org/licenses/by-nc/4.0/).

(B) Top: comparison between the cryo-EM maps of the Pol III PTC (left) and Pol III Δ (right). The Pol III Δ map lacks C53-C37 and C11. Bottom: the NT-strand binding region in the Pol III PTC. Cryo-EM densities are shown as transparent surfaces.

(C) Superimposition of the Pol III and Δ Pol III PTC structures. Conformational changes are indicated by red arrows.

(D) Superimposition of the Pol III and Δ Pol III EC structures.

of NTPs induced a register shift of the DNA:RNA hybrid but left the NT strand unchanged.

DISCUSSION

Model of Pol III transcription termination

We determined the high-resolution cryo-EM structure of the yeast Pol III PTC at a resolution of 2.8 Å. Using genetic, biochemical, and high-resolution genomic approaches, we validated the conclusions of our structural analyses *in vitro* and *in vivo*. We also determined complementary structures of the Pol III Δ and the Pol III PTC in the presence of NTPs. Based on these insights, we propose a mechanistic model for Pol III transcription termination (Figure 7).

Pol III initiates transcription with the help of GTFs (step 1). When Pol III enters the elongation phase, the NT strand only loosely associates with Pol III. Once Pol III reaches a termination site that hinders at least 4 dT bases, C128 recognizes the termination signal, Pol III pauses, and the Pol III PTC forms (step 2). PTC formation is driven by the H-bond network between C128 and the termination sequence. The NT strand becomes ordered, and Pol III contracts around it. FL 1 establishes multiple H bonds with the NT strand and forms a hydrophobic cavity that accommodates the methyl groups of 2 dT bases. C128-mediated recognition of the termination signal is aided by C53-C37, which binds the C128 lobe

domain. We earlier hypothesized that a flexible loop in C37 (aa 197-224), which connects two helices that bind the C128 lobe domain, participates in Pol III transcription termination by directly binding the NT strand (Hoffmann et al., 2015). Given that we did not observe such an interaction in the Pol III PTC, we need to revise this hypothesis and, instead, propose that binding of C53-C37 reduces the flexibility of C128 and, thereby, reinforces its tight interaction with the NT strand when the poly(dT) sequence is exposed. In the absence of C53-C37, Pol III would not be slowed down once it has reached the termination signal, thus preventing correct termination. This mode of function rationalizes the role of C53-C37 in transcription termination, which was, so far, poorly understood on a molecular level.

The interaction between the NT strand and C128 induces pausing, but Pol III still appears to be able to extend the RNA molecule, which “pulls” the T strand towards the active site (step 3). The strong interaction with the termination sequence hinders further translocation along the DNA and induces a register offset between the T and the NT strand. Such offset could trap Pol III in an unproductive state that might be sensed by subunit C11 (step 4). C11 is homologous to TFIIS, which is needed in the Pol II system to reactivate stalled Pol II by cleaving off backtracked RNA (Cheung and Cramer, 2011). We, therefore, speculate that C11 also senses an unproductive state of Pol III.

In attempts to rescue the stalled Pol III PTC, C11 may insert its catalytic C-terminal domain into the Pol III funnel. Insertion of C11 into the Pol III funnel has been recently shown to be concomitant with partial opening of the Pol III clamp domain that contacts the downstream DNA (Girbig et al., 2021) (step 5). However, transcription termination does not depend on the RNA-cleavage activity of C11 (Landrieux et al., 2006; Mishra and Maraia, 2019; Mishra et al., 2021). We, therefore, propose that C11 insertion into the Pol III funnel serves as a destabilizing signal by opening the clamp domain so that Pol III loses its “grip”
A

Pol III PTC + NTPs

<figure><img src="image_21.png" /></figure>

B

Pol III PTC + NTPs
DNA/RNA classified

<figure><img src="image_22.png" /></figure>

C

<figure><img src="image_23.png" /></figure>

Pol III PTC

<figure><img src="image_24.png" /></figure>

D

<figure><img src="image_25.png" /></figure>

Pol III PTC + NTPs

<figure><img src="image_26.png" /></figure>

E

<figure><img src="image_27.png" /></figure>

on the downstream DNA. Owing to the poly(dT) termination
signal on the NT strand, a dA:rU hybrid forms in the active site,
which is much less stable than hybrids with other sequences
(Martin and Tinoco, 1980) (step 6). The funnel insertion of C11
could reinforce dismantling of the already unstable hybrid. Since
the DNA:RNA hybrid itself is an important stabilizing element of
the Pol III EC, the falling apart of the hybrid would consequently
cause the Pol III PTC to collapse and to release both the DNA and
the nascent RNA molecule (step 7).

<caption>Figure 6. Structure of the Pol III PTC + NTPs</caption>

(A) Cryo-EM map of Pol III PTC + NTPs. Dark blue, T DNA; light blue, NT DNA; red, RNA.

(B) Improved signal for the upstream DNA, derived via masked classification on nucleic acid elements. A putative nucleic acid strand model for the upstream DNA is shown for illustrative purposes but was not included into the final Pol III PTC + NTPs model.

(C) Transcription bubble in the Pol III PTC. Nucleotides of interest are colored to illustrate the register of the DNA:RNA hybrid and of the NT strand.

(D) Transcription bubble in the Pol III PTC + NTPs structure. Nucleotides are colored as in (C). The nucleotide addition site (i + 1) shows weak density (green) for another added nucleotide.

(E) Schematic of the poly(dT) transcription bubble highlighting the observed register shift (arrows) in the DNA:RNA hybrid but not on the NT strand (dots) upon addition of NTPs.

C11 is also required for facilitated recy-
cling of Pol III (Dieci and Sentenac, 1996;
Landrieux et al., 2006). This mode of func-
tion could be assigned to the linker
domain that links the N-terminal and the
C-terminal domain (CTD) of C11 (Mishra
et al., 2021). We speculate that termi-
nated Pol III still harbors the C11 CTD in-
serted into its funnel and as an increased
propensity to reinitiate transcription (step
8). The cryo-EM structures of the yeast
Pol III initiation complexes revealed that
the clamp adopts a closed conformation
in the Pol III PIC (Abascal-Palacios et al.,
2018; Vorländer et al., 2018) and, thus,
needs to first open up to allow recruitment
of Pol III to the promoter DNA. We pro-
pose that this process could be assisted
by the insertion of C11 into the funnel.

Comparison between the yeast and
the human Pol III PTC

When this article was in preparation, the
structure of the human Pol III PTC at a res-
olution of 3.6 Å was reported (Hou et al.,
2021). The human Pol III PTC showed
characteristics comparable to those of
the yeast PTC (Figures S5A and S5B),

except that the DNA:RNA hybrid is more poorly resolved in the
human Pol III PTC (Figures S5C and S5D). The 6 bases of the pol-
y(dT) termination signal, however, were equally well resolved,
and multiple H bonds formed between the termination signal
and RPC2 (the human C128 ortholog). The NT strand follows a
similar path and is contacted by the FL 1 and FL 2
(Figures S5E and S5F). The lobe, protrusion, and fork elements
also contract around the NT strand. Together with our biochem-
ical investigation of the key residues in C128, our investigation of
<figure><img src="image_28.png" /><figcaption>**Figure 7. Model of Pol III transcription termination**</figcaption></figure>

The model combines the structural and biochemical data presented here with literature-based hypotheses (italicized).

in the two structures that might account for the observed differences, suggesting that the observed differences pertain to the impact of C11/RPC10. In both PTC structures, the CTDs of C11/RPC10 are positioned outside of the Pol III funnel. Human RPC10 is able to dynamically monitor the active site of Pol III because it adopts either an “outside funnel” (Girbig et al., 2021) or an “inside funnel” conformation (Girbig et al., 2021; Li et al., 2021; Wang et al., 2021). A similar conformation has not yet been captured for yeast Pol III, indicating that human RPC10 has a higher tendency to enter the Pol III active site. We hypothesize that the active site of human Pol III is, owing to the action of human RPC10, more sensitive to unstable poly(-dA:rU) hybrids than yeast Pol III, which could explain why human Pol III terminates transcription on fewer dTs.

## Limitations of the study

Our structural analysis reveals how Pol III recognizes and pauses on poly(dT) termination signals. Although we provide a possible model of the full Pol III termination process, it remains speculative until the precise role of the RNA-cleaving subunit C11 is deciphered, which will require additional structural and biochemical analysis. The Pol III PTC sample also did not contain a poly(dA:rU) hybrid to prevent

premature transcription termination. Thus, to what extent the formation and presence of the poly(dA:rU) hybrid affects Pol III transcription termination cannot be directly answered with our cryo-EM analysis, as it would require us to capture Pol III being bound to poly(dT) and a poly(dA:rU) hybrid. Capturing this state will likely prove challenging because Pol III may terminate before or during cryo-EM sample preparation. Nevertheless, such analysis will be an important next step to understand the Pol III transcription termination mechanism in full detail.

complementary structures, and the *in vivo* validation of its physiological relevance, the two Pol III PTC structures, thus, support and complement each other.

Notably, the number of dTs that are required for Pol III transcription termination differs between human and yeast. Whereas S. cerevisiae Pol III needs at least 5 dTs to terminate, the majority of human tRNA genes possess only a 4-dT termination site (Braglia et al., 2005), which is why human Pol III must be more sensitive to shorter dT-tracks. Given that the human and yeast Pol III PTCs recognize the NT strand in similar manners, we speculate that the differences between the two species are due to a less stably bound DNA:RNA hybrid in the active site, or to differences in the contribution of the C11 subunit (RPC10 in humans) to termination. Indeed, we noticed that the DNA:RNA hybrid in the human Pol III PTC was less well resolved than in the yeast Pol III PTC (Figures S5A–S5D). However, we could not observe any obvious differences in the amino acids that contact the DNA:RNA hybrids

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY
* Lead contact

* Materials availability

* Data and code availability

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* Yeast strains

* METHOD DETAILS

* Large scale purification of endogenous Pol III

* Small scale purification of endogenous Pol III

* Purification of yeast Pol III Δ

* Preparation of transcription scaffolds

* Cryo-EM sample preparation

* Cryo-EM data collection and processing

* Structural model building and refinement

* Residue conservation analysis

* Primer extension experiments

* *In vitro* transcription experiments

* Mutagenesis of *S. cerevisiae* C128 for structure-function studies

* Yeast spotting assays

* UV crosslinking and analysis of cDNA (CRAC)

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2022.111316.

## ACKNOWLEDGMENTS

We thank F. Weis and W.J.H. Hagen (EMBL Cryo-Electron Microscopy Service Platform) for EM support; T. Hoffmann and J. Pecar and EMBL IT Support for computational and data storage support, and T. Hoffmann and J. Pecar in particular for setting up and maintaining the high-performance computing environment; R.J. Maraia for sharing the yeast Pol III Δ expression strain; S.C. Vonesch for advice on CRISPR-Cas9-mediated genome engineering of yeast cells; F. Baudin for RNA labeling and advice on biochemical experiments; M.K. Vorländer for providing purified TFIIIB components and advice on cryo-EM data collection and processing; N.A. Hoffman for initial project discussions; and current members of the Müller lab for discussions. M.G., H.G., and C.W.M. acknowledge support from EMBL. M.G. was supported by a Boehringer Ingelheim Fonds PhD fellowship. D.L. and O.P. were supported by the Agence National pour la Recherche (ANR-16-CE12-0001-01 to O.P. and ANR-16-CE12-0022-01 to D.L.) and the Fondation pour la Recherche Medical (F.R.M., program Equipes 2019). J.X. was supported by the China Scholarship Council, the F.R.M. PhD fellowship (FDT20202102010433), and LabEx "Who Am I?" (ANR-11-LABX-0071 and Université de Paris IdEx ANR-18-IDEX-0001) funded by the French government through its "Investments for the Future."

## AUTHOR CONTRIBUTIONS

C.W.M. initiated and supervised the project. M.G. carried out protein purifications, biochemical experiments, cryo-EM grid preparation and data collections, and model building and interpreted the structures. H.G. performed genome engineering of yeast cells via CRISPR-Cas9, handled yeast cultures, and did yeast spotting assays. J.X. performed CRAC experiments. J.X., D.L., and O.P. analyzed and interpreted the CRAC results. M.G. wrote the manuscript with input from the other authors.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: March 10, 2022

Revised: July 1, 2022

Accepted: August 15, 2022

Published: September 6, 2022

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Zheng, S.Q., Palovcak, E., Armache, J.-P., Verba, K.A., Cheng, Y., and Agard, D.A. (2017). MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331-332.

Zivanov, J., Nakane, T., Forsberg, B.O., Kimanius, D., Hagen, W.J., Lindahl, E., and Scheres, S.H. (2018). New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>IgG from rabbit serum</td><td>Sigma-Aldrich</td><td>Cat# I5006; RRID: AB_1163659</td></tr><tr><td colspan="3">Bacterial and virus strains</td></tr><tr><td>Escherichia coli XL1-Blue chemically competent cells</td><td>Agilent</td><td>200249</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Tris</td><td>Sigma</td><td>T1503</td></tr><tr><td>HCl</td><td>Merck</td><td>1.00319</td></tr><tr><td>Ammonium sulfate</td><td>Applichem</td><td>A1032</td></tr><tr><td>Glycerol</td><td>Merck</td><td>1.04057</td></tr><tr><td>Lithium acetate (CH<sub>3</sub>COOLi)</td><td>Sigma</td><td>62393</td></tr><tr><td>EDTA</td><td>Merck</td><td>E5134</td></tr><tr><td>MgCl<sub>2</sub></td><td>Sigma</td><td>M9272</td></tr><tr><td>ZnCl<sub>2</sub></td><td>Sigma</td><td>39059</td></tr><tr><td>β-mercaptoethanol</td><td>Aldrich</td><td>M6250</td></tr><tr><td>DTT</td><td>Biomol</td><td>04010</td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>11017128001</td></tr><tr><td>Pepstatin A</td><td>Applichem</td><td>A2205</td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td></tr><tr><td>Benzamidin</td><td>Merck</td><td>12072</td></tr><tr><td>Hepes</td><td>Merck</td><td>H23830</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>56750</td></tr><tr><td>MgSO<sub>4</sub></td><td>Merck</td><td>S0505</td></tr><tr><td>Sodium acetate (CH<sub>3</sub>COONa)</td><td>Merck</td><td>236500</td></tr><tr><td>Urea</td><td>Merck</td><td>1.08487</td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.00983</td></tr><tr><td>Nourseothricin (clonNAT)</td><td>Jena Bioscience</td><td>AB-102L</td></tr><tr><td>Ampicillin</td><td>Applichem</td><td>A0839</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2438</td></tr><tr><td>Betaine</td><td>Sigma</td><td>B0300</td></tr><tr><td>PEG 4000</td><td>Merck</td><td>95904</td></tr><tr><td>Boric acid</td><td>Merck</td><td>B6768</td></tr><tr><td>Salmon sperm DNA</td><td>Thermofisher</td><td>15632011</td></tr><tr><td>NTP (ATP, CTP, GTP, UTP)</td><td>Thermofisher</td><td>R0481</td></tr><tr><td>[α-P32]UTP (10 mCi/ml)</td><td>Hartmann Analytic</td><td>FP-110</td></tr><tr><td>[γ-P32]ATP (10 mCi/mL)</td><td>Hartmann Analytic</td><td>FP-201</td></tr><tr><td>Formamide</td><td>Merck</td><td>F9037</td></tr><tr><td>Glycogen</td><td>ThermoFisher Scientific</td><td>10814010</td></tr><tr><td>Phenol-chloroform</td><td>Applichem</td><td>A0889</td></tr><tr><td>T4 PNK</td><td>Biolabs</td><td>M0201L</td></tr><tr><td>T7 ligase</td><td>Biolabs</td><td>M0318S</td></tr><tr><td>BsmBI</td><td>Thermofisher</td><td>ER0451</td></tr><tr><td>Phusion DNA Polymerase</td><td>Thermofisher</td><td>F530L</td></tr><tr><td>DpnI</td><td>ThermoScientific</td><td>FD1704</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Bradford reagent</td><td>ThermoScientific</td><td>23200</td></tr><tr><td>cOmplete EDTA-free protease inhibitor cocktail tablets</td><td>Sigma-Aldrich (Roche)</td><td>Cat# 11873580001</td></tr><tr><td>Dnase I recombinant, Rnase-free</td><td>Sigma-Aldrich (Roche)</td><td>Cat# 04716728001</td></tr><tr><td>Dynabeads M-280 Tosylactivated</td><td>Thermo Fisher Scientific</td><td>Cat# 14204</td></tr><tr><td>Recombinant GST-TEV protease</td><td>(Challal et al., 2018)</td><td>N/A</td></tr><tr><td>Rnace-It Ribonuclease Cocktail</td><td>Agilent</td><td>Cat# 400720</td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>Cat# G4505</td></tr><tr><td>Ni-NTA Agarose</td><td>QIAGEN</td><td>Cat# 30230</td></tr><tr><td>RNaseOUT Recombinant Ribonuclease Inhibitor</td><td>Thermo Fisher Scientific</td><td>Cat# 10777019</td></tr><tr><td>T4 RNA Ligase 2, truncated KQ</td><td>NEB</td><td>Cat# M0373L</td></tr><tr><td>T4 RNA Ligase 1 (ssRNA Ligase)</td><td>NEB</td><td>Cat# M0204L</td></tr><tr><td>Proteinase K, recombinant, PCR grade</td><td>Sigma-Aldrich (Roche)</td><td>Cat# 03115887001</td></tr><tr><td>SuperScript IV Reverse Transcriptase</td><td>Thermo Fisher Scientific</td><td>Cat# 18090050</td></tr><tr><td>Rnase H</td><td>NEB</td><td>Cat# M0297S</td></tr><tr><td>Exonuclease I</td><td>NEB</td><td>Cat# M0293S</td></tr><tr><td>LA Taq</td><td>Takara</td><td>Cat# RR002M</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>LightCycler 480 SYBR Green I Master</td><td>Roche</td><td>Cat# 04887352001</td></tr><tr><td>NucleoSpin Gel and PCR Clean-up</td><td>Macherey-Nagel</td><td>Cat# 740609</td></tr><tr><td>Pierce Spin Columns - Snap Cap</td><td>Thermo Fisher Scientific</td><td>Cat# 69725</td></tr><tr><td>Vivacon 500</td><td>Sartorius</td><td>Cat# VN01H22</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific (Invitrogen)</td><td>Cat# Q32851</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Fisher Scientific</td><td>Cat# 34080</td></tr></table>

Deposited data

<table><tr><td>Atomic coordinates of yeast Pol III PTC</td><td>This paper</td><td>PDB: 7Z1L</td></tr><tr><td>Cryo-EM map of yeast Pol III PTC (Map A)</td><td>This paper</td><td>EMDB: EMD-14447</td></tr><tr><td>Atomic coordinates of yeast Pol III EC</td><td>This paper</td><td>PDB: 7Z1M</td></tr><tr><td>Cryo-EM map of yeast Pol III EC (Map B)</td><td>This paper</td><td>EMDB: EMD-14448</td></tr><tr><td>Atomic coordinates of yeast Pol III Δ</td><td>This paper</td><td>PDB: 7Z1N</td></tr><tr><td>Cryo-EM map of yeast Pol III Δ (Map C)</td><td>This paper</td><td>EMDB: EMD-14449</td></tr><tr><td>Atomic coordinates of yeast Pol III PTC + NTPs</td><td>This paper</td><td>PDB: 7Z1O</td></tr><tr><td>Cryo-EM map of yeast Pol III PTC + NTPs (Map D)</td><td>This paper</td><td>EMDB: EMD-14451</td></tr><tr><td>Cryo-EM map of yeast Pol III PTC + NTPs 2 (Map E)</td><td>This paper</td><td>EMDB: EMD-14450</td></tr><tr><td>Genome-wide data</td><td>This paper</td><td>GEO: GSE196398</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>AC40-TAP-tagged S. cerevisiae strain to purify Pol III WT (large scale); genotype: SC0000; MATa; ade2; arg4; leu2-3,112; trp1-289; ura3-52; YPR110c::TAP-KIURA3</td><td>Cellzome GmbH</td><td>Euroscarf #SC1613</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III WT (small scale); genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c::TAP-KIURA3</td><td>Cellzome GmbH</td><td>Euroscarf #SC2330</td></tr></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>His<sub>6</sub>-FLAG<sub>3</sub>-C128-tagged S. cerevisiae strain to purify Pol III &Delta;; genotype: ret1::URA3MX, pRS313:N-His-FLAG- C128; rpo31D::KanMX, pRS313gu-nHA- C160, rpc11D::NatMX, ppRS316gu- spC11mvq</td><td>Maraia lab (NIH/NICHD, Bethesda), original citation: Kassavetis et al., 2010 (https://doi. org/10.1074/jbc.M109.074013)</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 triple mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_Q476A, K479A, R481G::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 Q199R mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_Q199R::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 R451V mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_R451V::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 K448A mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_K448A::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 R481G mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_R481G::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>C128-TAP-tagged S. cerevisiae strain to purify Pol III C128 H225L mutant; genotype: MATa; ade2; arg4; leu2-3,112 trp1-289; ura3-52; YOR207c_H225L::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>His<sub>6</sub>TAP-TRP1-tagged S. cerevisiae strain for CRAC, C128 WT; genotype: MATa, ade2, arg4, leu2-3, 112, trp1-289, ura3-52, YOR207c_WT, YOR116c::His<sub>6</sub>TAP-TRP1</td><td>This paper</td><td>N/A</td></tr><tr><td>His<sub>6</sub>TAP-TRP1-tagged S. cerevisiae strain for CRAC, C128 triple mutant; genotype: MATa, ade2, arg4, leu2-3, 112, trp1-289, ura3-52, YOR207c_Q476A, K479A, R481G::TAP-KIURA3</td><td>This paper</td><td>N/A</td></tr><tr><td>His<sub>6</sub>TAP-TRP1-tagged S. cerevisiae strain for CRAC, C128 Q199R mutant; genotype: MATa, ade2, arg4, leu2-3, 112, trp1-289, ura3-52, YOR207c_Q199R, YOR116c::His<sub>6</sub>TAP-TRP1</td><td>This paper</td><td>N/A</td></tr><tr><td>His<sub>6</sub>TAP-TRP1-tagged S. cerevisiae strain for CRAC, C128 R451V mutant; genotype: MATa, ade2, arg4, leu2-3, 112, trp1-289, ura3-52, YOR207c_R451V, YOR116c::His<sub>6</sub>TAP-TRP1</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Oligonucleotides are listed in Table S2.</td><td></td><td>N/A</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>Plasmid: pWS082</td><td>Addgene</td><td>90516</td></tr><tr><td>Plasmid: pWS174</td><td>Addgene</td><td>90961</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Software and algorithms</td></tr><tr><td>BEDTools</td><td>Quinlan and Hall, 2010</td><td>https://bedtools.readthedocs.io/en/latest/</td></tr><tr><td>Bowtie 2</td><td>Langmead and Salzberg, 2012</td><td>https://sourceforge.net/projects/bowtie-bio/files/bowtie2/</td></tr><tr><td>Coot 0.8.9.1</td><td>Emsley et al., 2010</td><td>https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td></tr><tr><td>cryoSPARC 2.15.0</td><td>Punjani et al., 2017</td><td>https://cryosparc.com/</td></tr><tr><td>deepTools</td><td>Ramírez et al., 2016</td><td>https://deeptools.readthedocs.io/en/develop/</td></tr><tr><td>FASTX-Toolkit</td><td>Hannon lab CSHL</td><td>http://hannonlab.cshl.edu/fastx_toolkit/</td></tr><tr><td>Image Lab</td><td>Bio-Rad</td><td>https://www.bio-rad.com/de-de/product/image-lab-software?ID=KRE6P5E8Z</td></tr><tr><td>ISOLDE 1.0</td><td>Croll, 2018</td><td>https://isolde.cimr.cam.ac.uk/</td></tr><tr><td>Jalview 2.11.0</td><td>Waterhouse et al., 2009</td><td>http://www.jalview.org/getdown/release/</td></tr><tr><td>LocalDeblur</td><td>Ramírez-Aportela et al., 2019</td><td>https://github.com/l2PC/xmipp</td></tr><tr><td>MonoRes</td><td>Vilas et al., 2018</td><td>https://github.com/l2PC/xmipp</td></tr><tr><td>pyCRAC</td><td>Webb et al., 2014</td><td>https://git.ecdf.ed.ac.uk/sgrannem/pycrac</td></tr><tr><td>PHENIX 1.19</td><td>Liebschner et al., 2019</td><td>https://phenix-online.org/</td></tr><tr><td>RELION 3.1</td><td>Zivanov et al., 2018</td><td>https://relion.readthedocs.io/en/release-3.1/</td></tr><tr><td>R 3.5.3</td><td>R Core Team</td><td>https://www.R-project.org</td></tr><tr><td>RStudio 1.1.456</td><td>RStudio Team</td><td>https://www.rstudio.com/</td></tr><tr><td>Scion 2.0</td><td>de la Rosa-Trevín et al., 2016</td><td>http://scion.cnb.csic.es/</td></tr><tr><td>SerialEM 3.8.0 beta</td><td>Mastronarde, 2005</td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>TeXshade 1.25</td><td>Beitz, 2000</td><td>https://ctan.net/macros/latex/contrib/texshade/</td></tr><tr><td>T-Coffee</td><td>Notredame et al., 2000</td><td>https://www.ebi.ac.uk/Tools/msa/tcoffee/</td></tr><tr><td>Trimmomatic</td><td>Bolger et al., 2014</td><td>http://www.usadellab.org/cms/index.php?page=trimmomatic</td></tr><tr><td>UCSF Chimera</td><td>Goddard et al., 2007</td><td>https://www.cgl.ucsf.edu/chimera/</td></tr><tr><td>UCSF ChimeraX</td><td>Goddard et al., 2018</td><td>https://www.cgl.ucsf.edu/chimerax/</td></tr><tr><td>Warp 1.0.6</td><td>Tegunov and Cramer, 2019</td><td>https://isolde.cimr.cam.ac.uk/</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christoph W. Müller (christoph.mueller@embl.de).

Materials availability

Plasmids and yeast strains are available upon request and will be available without restrictions.

Data and code availability

* Atomic coordinates and cryo-EM maps of the Pol III PTC, Pol III EC, Pol III Δ and Pol III PTC + NTPs have been deposited to the Protein Data Bank (PDB) and to the Electron Microscopy Data Base (EMDB) under the following accession codes: yeast Pol III PTC: PDB: 7Z1L, EMDB: EMD-14447; yeast Pol III EC: PDB: 7Z1M, EMDB: EMD-14448; yeast Pol III Δ: PDB: 7Z1N, EMDB: EMD-14449; yeast Pol III PTC + NTPs: PDB: 7Z1O, EMDB: EMD-14451; Pol III PTC + NTPs 2 (classified on nucleic acids): EMDB: EMD-14450. CRAC data has been deposited to the NCBI's Gene Expression Omnibus (GEO) under accession code GEO: GSE196398.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request
## EXPERIMENTAL MODEL AND SUBJECT DETAILS

### **Yeast strains**

Yeast cells were grown in YPAD, YPD, YPGAL or CSM-TRP medium and, if not otherwise stated, grown at a temperature of 30°C. Growth conditions for yeast cells strains varied between experiments and are, therefore, further specified in the *Method details* section. The genotypes for the used yeast strains can be found in the *Key resources table*.

## METHOD DETAILS

### **Large scale purification of endogenous Pol III**

Yeast Pol III was purified in large scale from a S. cerevisiae strain (Euroscarf ID #SC1613) carrying a C-terminal TAP-tag on subunit AC40. Large-scale purified Pol III was used for structural analysis and biochemical experiments. Yeast cells were grown in a 100 L fermenter (BIOSTAT® D-DCU, Sartorius) in YPAD medium for 17 h at 30°C. Cells were harvested in log phase (OD~600 nm~ > 6.5) with a flow-through centrifuge, which typically yielded a ca. 1.5 kg cell pellet that was kept at -80°C until usage. For purification, the yeast cell pellet was thawed over night at 4°C and, the next day, washed with cell wash buffer (50 mM Tris-HCl pH 8 (RT), 250 mM (NH~4~)~2~SO~4~, 12 mM β-mercaptoethanol) by dissolving the yeast pellet in 100 mL wash buffer per 100 g cell pellet and centrifugation (JLA 8.1000 rotor, Beckman) at 5,000 rpm, 4°C for 10 min. The supernatant was removed and the cell pellet was resuspended in lysis buffer (250 mM Tris-HCl pH 8 (RT), 20% glycerol, 250 mM (NH~4~)~2~SO~4~, 1 mM EDTA, 10 mM MgCl~2~, 10 µM ZnCl~2~, 12 mM mercaptoethanol), supplemented with protease inhibitor (PI) cocktail mix (0.3 µg/mL Leupeptin, 1.4 µg/mL Pepstatin, 170 µg/mL PMSF, 330 µg/mL benzamidin, in EtOH). For cell lysis, all material and equipment was pre-cooled at 4°C and lysis steps were performed at 4°C. Cells were lysed with glass beads in a bead beater (BioSpec) using ca. 230 mL cell suspension per beaker (ca. 6 beakers in total) and 170 mL glass beads per beaker, supplemented with a spatula tip of Pefabloc. Beakers were cooled using an ice-salty water (5 M NaCl) bath. Cells were lysed by beating 8 times for 45 s and waiting for 90 s. The lysate was filtered through a metal mesh and centrifuged (JA-14 rotor, Beckman) at 14,000 rpm, 4°C for 1 h. The supernatant was recovered, filtered again through a metal mesh and applied to a 350 mL heparin sepharose (GE Healthcare) column equilibrated in HEP bind buffer (50 mM Tris-HCl pH 8 (RT), 20% glycerol, 250 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 1 mM β-mercaptoethanol), supplemented with PI mix (see above). The remaining cell pellet (containing unlysed yeast cells) was resuspended in lysis buffer, lysed again using the procedure described above, and also applied to the heparin column. Bound proteins were eluted using a 400 mL linear gradient (0-100%) from buffer HEP A (50 mM Tris-HCl pH 8 (RT), 250 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 1 mM β-mercaptoethanol, PI mix) into HEP B (50 mM Tris-HCl pH 8 (RT), 1 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 1 mM β-mercaptoethanol, 0.5 mM PMSF). The eluate was diluted with HEP A buffer to 500 mM (NH~4~)~2~SO~4~ and incubated with 20 mL IgG sepharose beads (GE Healthcare), equilibrated by washing with first 0.5 M AcOH pH 3.4 and second 1M Tris-HCl pH 7.5 (RT) (repeated twice) 4°C, rolling over night. The next day, beads were washed with 200 mL IgG binding buffer (50 mM Tris-HCl pH 8, 10% glycerol, 225 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 2 mM β-mercaptoethanol), and bound proteins were eluted via addition of Tobacco etch virus (TEV) protease, incubation for 6 h at 4°C, and recovery of the supernatant. 100 mL of IgG elution buffer (50 mM Tris-HCl pH 8, 60 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 2 mM β-mercaptoethanol) were added to the IgG beads and the flow-through was recovered and pooled with the first eluate. The eluted protein solution was applied to a MonoQ 10/100 column (Sigma-Aldrich, Merck), equilibrated in MonoQ buffer A (40 mM Hepes, pH 7.5, 60 mM (NH~4~)~2~SO~4~, 0.5 mM EDTA, 1 mM MgCl~2~, 10 µM ZnCl~2~, 10 mM DTT). Bound proteins were eluted via linear gradient from 60 to 1000 mM (NH~4~)~2~SO~4~ in buffer and buffer exchanged into EM buffer (15 mM Hepes, pH 7.5, 150 mM (NH~4~)~2~SO~4~, 10 mM DTT). The protein sample was concentrated to 7.5-14.9 mg/mL, snap-frozen in liquid nitrogen and stored at -80°C until usage.

### **Small scale purification of endogenous Pol III**

Yeast Pol III was purified in small scale for structure-function studies from either a S. cerevisiae strain (Euroscarf ID #SC2330) carrying a C-terminal TAP-tag on subunit C128 (Pol III WT) or from S. cerevisiae mutant strains, genome-engineered via CRISPR-Cas9 (see below). Yeast cells were grown in 24 L cultures (in YPAD medium) in conical flasks. To do so, 30 mL of yeast over-night cultures were prepared and diluted to 100 mL the next morning. In the evening, the cultures were diluted to 500 mL. The next day, the cultures were diluted to an OD~600 nm~ of 0.2, split amongst 16 × 1.5 L cultures and grown to an OD~600 nm~ of 1.5. The cultures were harvested by centrifugation (JLA 8.1000 rotor, Beckman, 3000 rpm, 10 min), washed once with PBS, and snap frozen in liquid nitrogen. Frozen cells (ca. 50 g per 24 L) were thawed over night at 4°C and resuspended in lysis buffer (see above), supplemented with PI mix (see above). The cells were lysed as described above. The lysate was cleared via centrifugation and loaded on two 5 mL HiTrap Heparin HP (Cytiva, Merck) columns (connected in series) that were equilibrated in lysis buffer. Proteins were eluted using a 100 mL linear gradient (0.25 to 1 M (NH~4~)~2~SO~4~). Pol III was further purified as described above except that the MonoQ step was omitted. Buffer-exchanged proteins were concentrated up to 0.4 to 5.6 mg/mL, snap frozen, and stored at -80°C.

### **Purification of yeast Pol III Δ**

Pol III Δ (lacking C53/C37/C11) was purified for structural analysis from an S. cerevisiae strain harboring a His~6~-tag on subunit C128. The strain was a gift from Richard Maraia (National Institutes of Health, Bethesda, MD, USA) and features a substitution of
<figure><img src="image_29.png" /></figure>

endogenous S. cerevisiae C11 with S. pombe C11, which causes loss of the C53/C37 heterodimer upon protein purification (Kassavetis et al., 2010). Yeast cells were grown in a 100 L fermenter culture in YPGAL medium to an OD₆₀₀ nm of ca. 5.0–6.0. A 330 g pellet of frozen cells was thawed over night at 4°C and resuspended in lysis buffer (60 mM Hepes pH 7.5, 750 mM NaCl, 10.5 mM MgCl₂, 7.5% glycerol, 14.3 mM mercaptoethanol), supplemented with PI mix (see above). The cells were lysed as described above. The lysate was cleared via two centrifugation runs (1 h, 4°C, each): first run at 30,000 g; second run at 72,465 g. The lysate was loaded overnight onto a 5 mL HisTrap HP column (Cytiva, Merck) equilibrated in 20 mM Hepes pH 7.5, 500 mM NaCl, 7 mM MgCl₂, 10% glycerol, 10 mM mercaptoethanol, 10 mM imidazole). Bound proteins were eluted using a 100 mL linear gradient (10 to 300 mM imidazole), diluted with 120 mL of MonoQ buffer (40 mM Tris-HCl pH 8, 68 mM (NH₄)₂SO₄, 0.5 mM EDTA, 1 mM MgCl₂, 10 µM ZnCl₂, 10 mM DTT) and loaded onto a MonoQ 5/50 column (Sigma-Aldrich, Merck). Bound proteins were eluted using a 30 CV linear gradient (68 mM to 1 M (NH₄)₂SO₄), diluted with MonoQ A buffer, and applied a second time to the Mono Q 5/50 GL to increase purity. For the second elution step, a two-step linear gradient (first step: 68 mM to 250 mM (NH₄)₂SO₄; second step: 250 mM to 1 M (NH₄)₂SO₄) was used. Eluted proteins were buffer exchanged as described above, concentrated to 2.4 mg/mL, snap-frozen in liquid nitrogen and stored at -80°C until usage.

## Preparation of transcription scaffolds

Transcription scaffolds were always prepared freshly before usage. Scaffolds used for cryo-EM sample preparation were annealed in a thermocycler (Bio-Rad) in the following way: oligonucleotides were dissolved in water. 1 µL of the template-strand and 1 µL of the non-template strand (100 µM each) were mixed and incubated at 95°C for 3 min. The temperature was incrementally reduced to 25°C at a rate of 1°C/min and 3 µL of 2x hybridization buffer (2xHB) (40 mM Hepes pH 7.5, 24 mM MgCl₂, 200 mM NaCl, 20 mM DTT) were added. 1 µL of the RNA strand (100 µM) were pre-heated at 45°C for 3 min and added to the mixture, which was incubated at 45°C for 3 min. The temperature was reduced to 20°C at a rate of 0.7°C/min, and the annealed scaffold was kept on ice.

## Cryo-EM sample preparation

All samples were diluted to the working concentrations using EM buffer (15 mM Hepes, pH 7.5, 150 mM (NH₄)₂SO₄, 5 mM MgCl₂, 10 mM DTT), supplemented with 4 mM (final concentration) CHAPSO. For assembly of the nucleic-acid bound complexes, Pol III protein samples were incubated at 4°C for 60 min with 1.05x molar excess of pre-annealed transcription scaffolds (see above). The following final protein concentrations were used: 2.5 µM (Pol III PTC); 2.8 µM (Pol III EC); 3.4 µM (Pol III PTC + NTPs), 2.6 µM (Pol III Δ). For the Pol III PTC + NTPs sample, the EM buffer was further supplemented with CTP, ATP, and UTP (10 mM each) and the Pol III-nucleic acid complex was incubated for 90- instead of 60 min. After incubation with transcription scaffolds, 3 µL of sample was applied to 200-mesh copper R2/1 grids (Quantifoil), which were glow-discharged twice using a Pelco EasyGlow instrument. The cryo-EM grids were prepared at 100% humidity and 4°C using a Vitrobot Mark IV (Thermo Fisher Scientific) and the following blotting parameters: wait time 10 s, blot force 4, blot time 4 s. For cryo-EM sample preparation of Pol III Δ, a slightly different protocol was used: the DNA:RNA hybrid was first pre-annealed in a thermocycler by mixing 1 µL of T-DNA and 1 µL of RNA (100 µM each) using the same program as for the other annealing steps. After annealing, the DNA:RNA hybrid was diluted to a concentration of 12 µM in HB buffer (20 mM Hepes pH 7.5, 12 mM MgCl₂, 100 mM NaCl, 10 mM DTT). Next, 9 µL of Pol III Δ protein sample (c = 4 µM) were mixed with 3 µL DNA:RNA hybrid (c = 12 µM), and the mixture was incubated at 28°C for 10 min. Afterwards, 1 µL of NT-DNA (c = 40 µM, diluted in HB buffer) strand was added, and the reaction was incubated for another 10 min at 28°C before supplementing the mixture with 4 mM CHAPSO (final concentration) and plunge freezing as described above.

## Cryo-EM data collection and processing

SerialEM (Mastronarde, 2005) was used for automated data collection. Cryo-EM data were collected on a Titan Krios transmission electron microscope (TEM) operated at 300 keV (Thermo Fisher Scientific), equipped with a Quantum energy filter (Gatan), and with a K2 (for Pol III PTC, EC and PTC + NTPs) or K3 (for Pol III Δ) direct detector (Gatan). Cryo-EM data collection parameters (magnification, pixel size, number of collected movies, movie frames, exposure time, dose, defocus range) for the four datasets are specified in Table S1. Warp (Tegunov and Cramer, 2019) was used for initial frame-alignment, dose-weighting, estimation of the contrast transfer function (CTF) and particle picking. The data was further processed in RELION 3.1 (Zivánov et al., 2018). For particle polishing, frame-alignment and dose-weighting was also done via RELION's own implementation of the MotionCor2-algorithm (Zheng et al., 2017) and CTF-correction was done with Gctf (Zhang, 2016). CryoSPARC (Punjani et al., 2017) was also used at earlier stages of image processing to assess data quality via 2D classification and generation of a 3D ab-initio reconstruction of the Pol III PTC, which was later used as a reference for 3D classification in RELION. Particle numbers and data processing steps are outlined in Figures S2, S3, S5, and S6. Following global 3D classification (4–6 classes, T value = 4), selected particles were subjected to 3D auto-refinement. Derived 3D maps were used to create global masks, covering the entire Pol III molecules, which were used for RELION post-processing and subsequent masked 3D classification. Masked 3D classification was performed without image alignment, with two classes, and a T value of 20. Masked 3D classification using a global mask enabled separation of Pol III molecules with and without the C53-C37 heterodimer and, typically, yielded slightly improved resolution values for the classes containing the complete Pol III molecule (with C53-C37). In the case of the Pol III PTC, masked 3D classification was repeated using a mask that covered only the C53-C37 heterodimer. Subsequently, particle images were CTF-refined and subjected to particle polishing in RELION. For particle polishing, selected particle images were first re-extracted from 3D movies, pre-processed in RELION (see above), and subjected to 3D auto-refinement. Next, we subjected the selected particle images
to iterative CTF refinement using: i) beam tilt estimation, trefoil estimation, 4th order aberrations estimation, ii) anisotropic magnification estimation; and iii) CTF parameter fitting using ‘per particle’ defocus fitting and ‘per micrograph’ astigmatism fitting. For particle polishing, optimal parameters were first trained using 5000 particles. Polished particles were again subjected to 3D auto-refinement, following iterative CTF refinement performed as described above and a final 3D auto-refinement run, which yielded the final 3D reconstructions used for map sharpening and atomic model building. In case of the Pol III PTC + NTPs dataset, the region corresponding to the upstream DNA was better resolved than in the other 3D reconstructions. We, therefore, performed masked 3D classification on the bound nucleic acids, which yielded a map that featured a clearly visible upstream DNA moiety (map E). Reported nominal resolution values of 3D reconstructions were calculated via RELION post-processing using the Fourier shell correlation (FSC) 0.143 cut-off criterion (Rosenthal and Henderson, 2003). Local resolution ranges were estimated using RELION.

## Structural model building and refinement

Cryo-EM maps, used for structural model building, were sharpened via Local Deblur (Ramírez-Aportela et al., 2019), implemented into the Scion software framework (de la Rosa-Trevín et al., 2016). Scion, in turn, was run via the SBGrid software collection (Morin et al., 2013). Structural model building was performed using Coot (Casañal et al., 2020; Emsley et al., 2010). Starting models were placed into cryo-EM density maps using UCSF Chimera (Goddard et al., 2007). The first map used for structural model building was of the Pol III PTC. The model of the yeast Pol III EC (Hoffmann et al., 2015) was used as the starting model (PDB: 5FJ8). The model was first real-space refined in Coot using distance restraints to accommodate for conformational changes of the newly build model. Distance restraints were computed using ProSMART (Nicholls et al., 2012), implemented into Coot. The sequence of the DNA:RNA scaffold was mutated to resemble the scaffold used in the Pol III PTC sample. Clear additional density could be observed of the NT-strand, and the sequence of the poly-dT termination signal was manually build into this density. The model was further manually adjusted without distance restraints to fit the cryo-EM density of the Pol III PTC and real-space refined in Coot using Ramachandran restraints. The Pol III PTC model was further subjected to ISOLDE (Croll, 2018) to reduce the clash score and optimize the model geometry. For the Pol III EC structure, the Pol III PTC model was used as a starting model. Conformational changes were first adjusted by subjecting the Pol III PTC model and the Pol III EC map to the Namdinator software (Kidmose et al., 2019). The sequence of the nucleic acids was adjusted to resemble the used EC scaffold. No density of the unwound NT-strand could be observed, and the corresponding region was deleted. The Pol III EC model was further manually inspected and adjusted in Coot. For the Pol III Δ model, the procedure was the same as for the Pol III EC except that the C53/C37 heterodimer and C11 were deleted from the Pol III PTC starting model. For the Pol PTC + NTPs structure, a register shift of the DNA:RNA hybrid could be observed when inspecting the density in the active site, and the DNA:RNA hybrid was adjusted accordingly. Weak density for a nucleotide base at the position i+1 was also noticed and the 3'-end of the RNA was, therefore, extended with an adenosine monophosphate molecule. Interpretation of the upstream DNA in map E was further aided by subjecting map E to LocScale (Jakobi et al., 2017). All models were refined multiple times against the corresponding cryo-EM maps with Phenix (Liebschner et al., 2019) using Ramachandran- and secondary structure restraints. ChimeraX (Goddard et al., 2018) was used to visualize cryo-EM maps and atomic models for the structural analysis and interpretation and for figure preparation.

## Residue conservation analysis

To analyze the conservation of C128 residues that contact the NT-strand, we performed a multiple-sequence alignment (MSA) of C128 orthologs from 15 eukaryotic species covering a wide range of the eukaryotic tree of life (Keeling and Burki, 2019): ophistokonts (S. cerevisiae, S. pombe, C. elegans, H. sapiens, A. queenslandica, C. owczarzaki, S. arctica), stramenopiles (T. pseudonana, N. gaditana); alveolata (P. falciparum, T. thermophila); archaeoplastids (P. patens, A. thaliana, C. merolae); amoebozoans (D. discoideum). Next, we subjected the MSA to the Scorecons software (Valdar, 2002) to retrieve conservation scores, which were used to color-code the NT-strand-binding residues in a nucleotide-plot.

## Primer extension experiments

For assembly of scaffolds used for primer extension experiments, the DNA and RNA oligonucleotides (1 µL, 20 µM each) were mixed and incubated at 95°C for 3 min. The mixture was placed immediately on ice for 1 min. Afterwards, 3 µL of 2x HB buffer were added, and the mixture was incubated at room temperature for at least 10 min. Prior to scaffold assembly, the used RNA was radioactively labelled on the 5'-end with [γ-32P]ATP (10 mCi/mL, Hartmann Analytic) and T4 PNK (NEB), following purification via PAGE. The annealed scaffold was diluted to 2 µM with transcription reaction buffer (TRB) (20 mM Hepes, pH 7.6, 60 mM (NH₄)₂SO₄, 10 mM MgSO₄, 10% glycerol, 2 mM DTT). 1 µL of diluted scaffold was incubated with 1 µL of Pol III (4 µM, also diluted in TRB) for at least 10 min at RT. Primer extension was initiated via addition of 7 µL TRB, supplemented with NTPs (1 mM each, final concentration). The reaction was incubated at 28°C for 20 min and stopped by adding 3 µL of formamide and boiling for 4 min. The reaction was subjected to PAGE using a denaturing (8M Urea) 17% polyacrylamide TBE gel. Radioactive signal was captured with a phosphor-imaging screen (Fujifilm) and detected with a Typhon FLA9500 (GE Healthcare) instrument.

## In vitro transcription experiments

Promoter-dependent and independent were performed to analyse Pol III transcription termination. Annealing of the DNA oligonucleotides (Sigma-Aldrich, desalt grade) was done similarly for the two types of experiments: 1 µL of template DNA-strand (30 µM) and
1 µL of non-template DNA-strand (30 µM) were mixed, boiled for 2 min, and immediately placed on ice for 1 min 2 µL of 2x HB (see above) were added, and the mixture was incubated at room temperature for 10 min. The annealed DNA was diluted to a concentration of 2 µM using TRB (see above) and kept on ice until usage. For promoter-independent experiments, tailed-template DNA constructs were designed to carry a single-stranded DNA overhang on the template strand. In a 5 µL reaction volume, 2 pmol of DNA were mixed with 2.5 pmol of WT or mutant Pol III, which were pre-diluted to working concentrations (0.6–2.5 µM) using EM buffer. The mixture was incubated at room temperature for 10 min, and transcription was initiated by adding 5.3 µL of a transcription initiation mix (3.9 µL of TRB, 0.7 µL NTP mix (10 mM ATP, 10 mM CTP, 1 mM UTP) and 0.7 µL of [α-P32]UTP (10 mCi/mL)). The reactions were incubated at room temperature for 40 min. Afterwards, 138 µL of stop buffer (0.6 M NaAcetate, 30 mM EDTA, 0.5% SDS) were added, and the reaction was subjected to phenol-chloroform extraction, boiling for 3 min, and denaturing PAGE using a 15% polyacrylamide TBE-Urea gel. Radioactive signal was recorded as described above. For promoter-dependent transcription assays, a double-stranded (ds) DNA hybrid construct was designed that consisted of the S. cerevisiae U6 snRNA promoter region and a portion of the S. cerevisiae tDNA gene tL(CAA)L featuring a 5-dT termination signal. Nucleic acids were annealed as described above. Pol III was diluted to 5 µM using TRB. Yeast TFIIIB, which was used to recruit Pol III to the promoter region, was freshly assembled by mixing a fusion construct of Brf1 and TBP (Brf1-TBP) with Bdp1, which were expressed and purified as described in (Vorländer et al., 2018). TFIIIB was assembled by mixing 2.4 µL of Bdp1 (6 mg/mL), 2.8 µL of Brf1-TBP (6 mg/mL) and 4.8 µL of TRB-300 (20 mM Hepes, pH 7.6, 300 mM NaCl, 60 mM (NH₄)₂SO₄, 10 mM MgSO₄, 10% glycerol, 2 mM DTT). The mixture was incubated on ice for 10 min and diluted to 5 µM using TRB-300. Next, 5 pmol of assembled TBIIIB were incubated with 2.5 pmol of dsDNA on ice for 10 min. Subsequently, 5 pmol of Pol III were added and the mixture was incubated for at least 10 min on ice. Transcription was initiated by adding 5.4 µL of transcription initiation mix (3.9 µL of TRB, 0.7 µL NTP mix (10 mM ATP, 10 mM CTP, 10 mM GTP, 1 mM UTP) and 0.7 µL of [α-P32]UTP (10 mCi/mL)). The reaction was incubated at 28°C. Typically, time courses were performed by stopping the reaction at the desired time points with 4 µL formamide loading buffer and boiling at 3 min. The reactions were directly loaded on 15% denaturing PAGE and radioactive transcription products were recorded as described above. The here-shown gels derived from promoter-dependent transcription experiments were not performed in triplicates but the reproducibility could be confirmed by performing the experiments multiple times for different analytical purposes.

## Mutagenesis of S. cerevisiae C128 for structure-function studies

CRISPR-Cas9-mediated genome engineering was used for site-directed mutagenesis of the endogenous S. cerevisiae C128-coding gene (RET1). A C128-TAP-tagged yeast strain (Euroscarf ID: SC2330) was used for genome engineering. Guide RNAs (sgRNA) were designed using the Benchling CRISPR Guide RNA Design tool (2021), retrieved from https://benchling.com, and ordered as DNA oligonucleotides in forward and reverse directions. For cloning of the sgRNA constructs, the oligonucleotides were phosphorylated via T4 PNK (NEB), mixed, and annealed by incubating the mixture at 95°C for 6 min and letting it cooling down to room temperature. Next, 2 µL of the annealed dsDNA was mixed with 0.5 µL of the pWS082 entry vector (which was a gift from Tom Ellis lab and Addgene plasmid # 90516) (at a concentration of 200–300 ng/uL), 1 µL each of T4 ligase, 10× T4 ligase buffer, and Bmsbl. In a thermocycler, the mixture was incubated at 42°C (2 min) and 16°C (5 min), and the cycle was repeated 10 times. Next, the reaction was incubated at 60°C for 10 min and lastly at 80°C for 10 min. The reaction was transformed into E. coli XL-1 Blue chemically competent cells (Agilent). The next day, positive clones were selected by using a Typhoon imager with a GFP-filter to screen for GFP-negative colonies. DNA donor constructs (containing the mutated DNA sequence to generate the corresponding C128 mutation) were designed to contain 50 base pairs (bps) homology arms at either side of the desired DNA break. The oligonucleotides were ordered as two partially complementary (20 bps) primers in forward and reverse direction, amplified via polymerase chain reaction (PCR), and purified via ethanol precipitation. The Cas9-sgRNA gap repair vector pWS174 (which was a gift from Tom Ellis lab and Addgene plasmid # 90961) was linearized with BsmBI (NEB) and gel-purified. The cloned sgRNA entry vectors were digested with EcoRV. For transformation into yeast cells, 5 µg of dsDNA donor DNA, 100 ng of linearized pWS174, and 200 ng of linearized sgRNA entry vector were mixed and transformed into yeast cells via the lithium acetate (LiAc)/sorbitol method. 50 mL of yeast cells at an OD₆₀₀ nm of 0.6 were spun down (2500 rpm, 5 min RT), washed once with a mix of LiAc (1M)/Sorbitol (1M) and resuspended in 600 µL LiAC/Sorbitol. The DNA mixture (above) was added to 100 µL of the yeast LiAc/Sorbitol suspension, and the mixture was added to a pre-mix containing 280 µL of 50% PEG 4000 and 15 µL sperm carrier DNA. The reaction was incubated at RT for 1 h, 40 µL of DMSO were added, and the reaction was incubated at 42°C for 20 min while slowly shaking. Next, 1 mL of YPD were added, followed by centrifugation (3000 rpm, 2 min). The pellet was dissolved in 500 µL YPD, transferred into 15 mL tubes containing 4.5 mL YPD, and the mixture was incubated at 30°C for at least 3 h. The cell suspension was spun at 2300 rpm for 5 min, dissolved in 500 µL PBS, spread on YPD plates supplemented with Nourseothricin (clonNAT) (100 ug/mL), and kept at 30°C. After three days, yeast colonies were re-streaked on YPD plates without clonNAT and incubated for another two or three days. Positive clones (those who contain the desired mutation) were screened via colony PCR and subsequent Sanger sequencing.

## Yeast spotting assays

YPD medium was inoculated with yeast cultures, and the yeast starter cultures were grown over night at 30°C shaking. The next day, the OD₆₀₀ nm was measured, the cultures were diluted to an OD₆₀₀ nm of 0.2 using fresh YPD, and the cultures were incubated for another 4 h. Next, the cultures were diluted to OD₆₀₀ nm = 0.2 using PBS. The PBD-diluted cell suspensions were used to make
10-fold serial dilutions in PBS (3 steps). Starting with the OD<sub>600 nm</sub> 0.2 dilutions, the diluted cultures were applied onto YPAD plates as single 8 µL drops. The plates were incubated at 25, 30 or 37°C and imaged after 3 days.

## UV crosslinking and analysis of cDNA (CRAC)

CRAC was performed using Pol III C128 WT and mutants yeast strains that carried an HTP-tag on subunit C160. The TAP-tag of C128 was, thus, removed for all three tested mutant yeast strains and replaced with a HTP-tag on C160 via homologous recombination. The CRAC protocol used in this study is derived from (Granneman et al., 2009) and modified as previously described (Candelli et al., 2018). Briefly, 2 L of yeast cells expressing an HTP-tagged version of C160 were grown at 30°C to OD<sub>600 nm</sub> 0.4–0.6 in CSM-TRP medium. Cells were crosslinked for 50 s using a W5 UV crosslinking unit (UVO3 Ltd) and harvested by centrifugation. Cell pellets were washed once with cold 1xPBS and resuspended in 2.4 mL/g of cells) TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40 and 5 mM β-mercaptoethanol) in the presence of protease inhibitors (Complete™ EDTA-free Protease Inhibitor Cocktail, Roche). Suspensions were flash frozen in droplets and cells subjected to cryogenic grinding using a Ball Mill MM 400 (5 cycles of 3 min at 20 Hz). The resulting frozen cell lysates were thawed on ice, digested with DNase I (165 units per gram of cells) at 25°C for 1h, and then clarified by centrifugation at 13,000 rpm for 30 min at 4°C. Protein extracts were quantified by Bradford reagent (Sigma) and the same amount of extracts for each sample were subjected to IgG affinity purification as described below.

RNA-protein complexes were bound on M-280 tosylactivated dynabeads coupled with rabbit IgGs (10 mg of beads per sample), washed with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% NP-40 and 5 mM β-mercaptoethanol), and eluted by TEV protease digestion. RNAs in the eluates were shortened by treating with 0.2 U of RNase cocktail (RNace-IT, Agilent) and the reaction was stopped by adding of guanidine–HCl to a final concentration of 6 M. The eluates were then incubated with Ni-NTA sepharose (Qiagen, 100 µL of slurry per sample) at 4°C overnight and extensively washed. Sequencing adaptors were ligated to the RNA molecules as described in Granneman et al. (2009). RNA-protein complexes were recovered with elution buffer (50 mM Tris pH 7.8, 50 mM NaCl, 150 mM imidazole, 0.1% NP-40 and 5 mM β-mercaptoethanol) and fractionated by Gel Elution Liquid Fraction Entrapment Electrophoresis (GelFree) system (Expedeon) according to manufacturer's instructions. The fractions containing[R]pc160 were digested with 100 µg of proteinase K, and RNAs were purified and reverse-transcribed using reverse transcriptase Superscript IV (Invitrogen).

The resulting cDNAs were amplified by PCR using LA Taq polymerase (Takara), and the PCR reactions were then treated with 200 U/mL of Exonuclease I (NEB) for 1 h at 37°C. The final library was purified using NucleoSpin columns (Macherey-Nagel) and sequenced on a NextSeq 500 Illumina sequencer.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Cryo-EM data were processed with RELION and cryoSPARC. Structural models were refined with Phenix. The corresponding statistics can be found in Table S1 and Data S1–S4.

For quantification and analysis of *in vitro* transcription experiments, RNA band intensities were quantified via the Image Lab software (Bio-Rad). Bar plots were generated in R. To normalize the radioactive signals of the bands of interest, their band intensities were divided by total lane intensities that contained the respective band of interest. The calculated values were multiplied by a factor of thousand for illustrative purposes. Tailed-template experiments were performed in triplicates (N = 3, technical replicates), except for the mutant K448A on the 3-dT construct, which was done in duplicates (N = 2, technical replicates). Mean values and standard deviations were calculated in R.

For CRAC experiments, two technical replicates were performed and were processed and statistically analysed as follows:

For CRAC data processing, the sequencing dataset was processed as described in (Xie et al., 2022) with some modification. Briefly, CRAC reads were demultiplexed using the pyBarcodeFilter script from the pyCRAC utility suite (Webb et al., 2014). The 5' adaptor was clipped with Cutadapt and the resulting insert quality-trimmed from the 3' end using Trimmomatic rolling mean clipping (Bolger et al., 2014). PCR duplicates were collapsed by the pyCRAC script pyFastqDuplicateRemover using a 6-nucleotide random tag included in the 3' adaptor. The resulting sequences were reverse complemented with the Fastx reverse complement that is included in the fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and mapped to the R64 genome with bowtie2 using "-N 1" option. Reads shorter than 20 nt were filtered out after mapping. Reads mapped to 37S rDNA unit (transcribed by Pol I) were also excluded from analysis to reduce the background signals. Reads mapping to multiple sites (e.g. most reads mapping to mature tRNA regions) were attributed randomly to one of the matching sites. Coverage bigwig files were generated and normalized to counts per million (CPM) using the bamCoverage tool from the deepTools package (Ramirez et al., 2016) using a bin size of 1.

For bioinformatic analysis, yeast genome was obtained from Saccharomyces Genome Database (S. cerevisiae) genome version R64-2-1). tRNA coordinate files (from 5' end to the end of the primary terminator) were obtained from (Xie et al., 2022). For each tRNA gene, the primary terminator was defined as the 1st T-tract after the 3' end of the mature tRNA. Four pairs of tRNAs that are arranged in tandem were excluded from the following analysis.

Reads mapped to different classes of RNAs were summarized by BEDTools coverage. Coverage files were analyzed with deepTools suite (Ramírez et al., 2016). Specifically, for metagene analysis of Pol III occupancy, strand-specific bigwig files and tRNA coordinate files were used as inputs for the computeMatrix tool using a bin size of 1 and the scale-regions mode. The matrices generated were then combined by the computeMatrixOperations tool with the rbind option and used as inputs for the plotProfile tool. For heatmap analyses, the log2 ratio of the Pol III signal in the indicated mutants relative to the WT was calculated by the bigwigCompare
<figure><img src="image_30.png" /></figure>

tool using a bin size of 1, and the resulting files were used as inputs for the computeMatrix as described above. The final matrix after
combination was used as inputs for the plotHeatmap tool. To analyze the correlation between two replicates, the average Pol III signal
over regions comprising tRNA genes and 500 bp upstream and downstream regions was computed using the multiBigwigSummary
tool. The signals calculated were then compared by plotCorrelation tool to generate scatter plots and compute the correlation co-
efficients using the Spearman method.

The efficiency of transcription termination in the WT and the mutants was estimated by calculating the read-through index (RT in-
dex) defined as the percentage of Pol III signal over the read-through regions (500 bp region immediately downstream of the primary
terminator of each tRNA gene) relative to the signal over tRNA gene regions (from 5' end to the end of the primary terminator). The
total Pol III signal at each region was computed with the UCSC bigWigAverageOverBed package (http://genome.ucsc.edu).

Data representation and statistical analyses were performed with R using the ggpubr (https://rpkgs.datanovia.com/ggpubr/) and
dplyr (https://dplyr.tidyverse.org/) packages.
Supplemental information

Architecture of the yeast Pol III
pre-termination complex and pausing mechanism
on poly(dT) termination signals

Mathias Girbig, Juanjuan Xie, Helga Grötsch, Domenico Libri, Odil Porrua, and Christoph W. Müller
<figure><img src="image_31.png" /><figcaption>Figure S1. Pol III terminates transcription on poly-dT sequences *in vitro*, Related to Figure 1.</figcaption></figure>

(A) Schematic of the *S. cerevisiae* dsDNA construct, containing the U6 snRNA promoter and the 3'-flanking site of the L(CAA)LR2 gene. The construct was used for promoter-dependent transcription experiments. The termination signal is highlighted in bold letters.

(B) Denaturing RNA gel of the promoter-dependent transcription experiment. Shown is a time course of probes taken at the indicated time points. A radioactive RNA product becomes visible over time that is labelled with an arrow; n (dsDNA) = 2.5 pmol; n (Pol III) = 5 pmol; n (TFIIIB) = 5 pmol. 15% TBE denaturing PAGE. The gels were cropped for clarity.

(C) Time-course of the Pol III transcription experiment testing the effect of a 5-dT, 4-dT, or 3-dT sequence on Pol III transcription termination. Same conditions as in (B). The gel has been cropped for clarity.
<figure><img src="image_32.png" /><figcaption>Figure S2. Cryo-EM data quality of the active site elements and newly build regions, Related to Figure 1.</figcaption></figure>

(A) Close-up views on the Pol III PTC active site elements. Cryo-EM densities are shown as transparent surfaces.

(B) The C11 C-terminal domain is visible and binds the Pol III core close to the C160 jaw domain and subunit ABC27.

(C) Newly built region (101-161) of the Pol III heterotrimer subunit C31. Cryo-EM density of the newly assigned region is shown as a transparent surface.

(D) Newly built region (195-227) of the Pol III heterodimer subunit C53. Cryo-EM density of the newly assigned region is shown as a transparent surface.

(E) - (F) Cryo-EM densities of putative CHAPSO molecules, of which the tetracyclic sterol head groups were placed.
<figure><img src="image_33.png" /><figcaption>Figure S3. NT-strand exit channel comparison between yeast Pol III PTC and yeast Pol I and Pol II EC structures, Related to Figures 1, 2.</figcaption></figure>

(A) Superimposition of the Pol III PTC and the Pol I EC (PDB: 6HLR). The C128 fork, protrusion, and lobe domains are labelled.

(B) Superimposition of the Pol III PTC and the Pol II EC (PDB: 5C4J).

(C) Calculated surface representations of C128 (Pol III PTC, left), A135 (Pol I EC, middle), and Rpb2 (Pol II EC, right).
<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

Replicate 2

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /><figcaption>Figure S4. Complementary analyses validating CRAC experiments, Related to Figure 4.</figcaption></figure>

A) Comparison of the read distribution among different genomic regions in the indicated strains in our Pol III CRAC experiment. The "others" category corresponds to Pol II genes and intergenic regions. Note that we have more contaminants and lower signal at tRNA regions in the WT_r1 and Triple_r1
samples, which, however, does not significantly affect the assessment of the transcription termination efficiency.

B) Plot representing the number of mapped reads obtained in a typical CRAC experiment in the different samples. "M" denotes millions.

C) Scatter plots showing the high correlation, calculated by the Spearman method, between the two biological replicates of each strain for the CRAC experiments showed in figure 4.

D) Integrative Genomics Viewer (IGV) screenshots of examples of tRNA genes displaying termination defects in the two biological replicates of each strain. The values under brackets correspond to the scale of the Pol III signal expressed in CPM. Note that because of the higher amounts of contaminants in the WT_r1 and Triple_r1 samples, the signal in the tRNA regions is lower (scale indicated in red), but the Pol III distribution is similar as in the other replicate of the same strains (r2).

E) IGV screenshots of RNAPIII-dependent genes other than tRNA genes presenting termination defects in RNAPIII mutants. Images are zoom in views of the corresponding termination regions.
<figure><img src="image_45.png" /><figcaption>B Human Pol III PTC
(Hou et al.)</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>Figure S5. Comparison between yeast and human Pol III PTC, Related to Figures 1, 2.</figcaption></figure>

(A) Cryo-EM map (unsharpened) of the yeast Pol III PTC (map A). Colour scheme: Pol III – grey, RNA – red, template (T)-DNA – dark blue, non-template (NT)-DNA – light blue.

(B) Cryo-EM map of the human Pol III PTC (EMD-31622; Hou et al., 2021). Colour scheme as in (A).

(C) Close-up view onto the active site of the yeast Pol III PTC (map A, unsharpened). Nucleic acid elements are coloured as in (A) and are labelled, accordingly. The ‘i-1’ refers to the nucleotide position adjacent to the nucleotide (i) addition site and marks the 3’-end of the RNA. ‘i-9’ marks the last RNA base that could build with high confidence. Threshold level is similar as in (A).

(D) Close-up view onto the active site of the human Pol III PTC (EMD-31622; Hou et al., 2021). Nucleic acid elements are coloured as in (C). Shown is the supplementary (derived from focused refinement onto the Pol III core) cryo-EM map of EMD-31622. Cryo-EM densities are shown at two
threshold levels (left one is similar as in (B)) to illustrate that the RNA in the human Pol III PTC is only weekly bound and can only be observed at lower cryo-EM threshold levels.

(E) Close-up views onto the fork regions of the yeast (y) (left) and human (h) (right) PTC structures and corresponding cryo-EM maps (in grey). The fork loop (FL) 1 and 2 elements (yeast – purple, human – burly-wood) and the NT-strands (yeast – light blue, human – brown) are coloured and labelled. Only subunits C128 (yeast) and RPC2 (human) are shown.

(F) Close-up view onto the Pol III fork region and overlay between atomic models of the yeast (solid ribbon model) and human Pol III (transparent ribbon model) PTC. Models are coloured as in (E).
<table><caption>Table S1. Cryo-EM data collection, refinement and validation statistics, Related to Figures 1, 2, 5, 6 and Data S1-S4</caption><thead><tr><th></th><th>Pol III<br>PTC<br>Map A</th><th>Pol III<br>EC<br>Map B</th><th>Pol III<br>Δ<br>Map C</th><th>Pol III PTC<br>+ NTPs<br>Map D</th><th>Pol III PTC<br>+ NTPs 2<br>Map E</th></tr></thead><tbody><tr><td>PDB code</td><td>7Z1L</td><td>7Z1M</td><td>7Z1N</td><td>7Z1O</td><td>-</td></tr><tr><td>EMDB code</td><td>EMD-14447</td><td>EMD-14448</td><td>EMD-14449</td><td>EMD-14451</td><td>EMD-14450</td></tr><tr><td colspan="6"><strong>Data collection and<br>processing</strong></td></tr><tr><td>Magnification</td><td>130,000</td><td>130,000</td><td>81,000</td><td>130,000</td><td>130,000</td></tr><tr><td>Voltage (kV)</td><td>300</td><td>300</td><td>300</td><td>300</td><td>300</td></tr><tr><td>Electron exposure (e-/Å<sup>2</sup>)</td><td>40.9</td><td>38.1</td><td>39.6</td><td>44.8</td><td>44.8</td></tr><tr><td>Defocus range (μm)</td><td>0.75-2.25</td><td>0.50-1.75</td><td>1.00-2.50</td><td>0.75-2.25</td><td>0.75-2.25</td></tr><tr><td>Pixel size (Å)</td><td>1.041</td><td>1.040</td><td>1.053</td><td>1.041</td><td>1.041</td></tr><tr><td>Symmetry imposed</td><td>C1</td><td>C1</td><td>C1</td><td>C1</td><td>C1</td></tr><tr><td>Initial particle images (no.)</td><td>464,321</td><td>248,884</td><td>359,678</td><td>538,161</td><td>538,161</td></tr><tr><td>Final particle images (no.)</td><td>47,222</td><td>24,295</td><td>18,530</td><td>237,468</td><td>23,641</td></tr><tr><td>Map resolution (Å)</td><td>2.8</td><td>3.4</td><td>3.9</td><td>2.7</td><td>3.2</td></tr><tr><td>FSC threshold</td><td>0.143</td><td>0.143</td><td>0.143</td><td>0.143</td><td>0.143</td></tr><tr><td>Map resolution range (Å)</td><td>2.5-8.0</td><td>3.2-8.8</td><td>3.7-8.5</td><td>2.5-5.1</td><td>2.9-7.5</td></tr><tr><td colspan="6"><strong>Refinement</strong></td></tr><tr><td>Initial model used (PDB code)</td><td>6TUT, 5FJ8,<br>5FJA</td><td>6TUT, 5FJ8,<br>5FJA</td><td>6TUT, 5FJ8,<br>5FJA</td><td>6TUT, 5FJ8,<br>5FJA</td><td></td></tr><tr><td>Model resolution (Å)</td><td>3.0</td><td>3.4</td><td>3.9</td><td>3.0</td><td></td></tr><tr><td>FSC threshold</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.5</td><td></td></tr><tr><td>Map sharpening B factor (Å<sup>2</sup>)</td><td>-27.6</td><td>-70.4</td><td>-107.1</td><td>-66.4</td><td></td></tr><tr><td colspan="6"><strong>Model composition</strong></td></tr><tr><td>Non-hydrogen atoms</td><td>42,330</td><td>42,127</td><td>38,605</td><td>42,375</td><td></td></tr><tr><td>Protein/nucleotide residues</td><td>5156/63</td><td>5153/53</td><td>4705/56</td><td>5154/66</td><td></td></tr><tr><td>Ligands</td><td>5x Zn, 1x Mg,<br>2x 1N7</td><td>5x Zn, 1x Mg,<br>2x 1N7</td><td>5x Zn, 1x Mg,<br>2x 1N7</td><td>5x Zn, 1x Mg,<br>2x 1N7</td><td></td></tr><tr><td colspan="6"><strong>B factors (Å<sup>2</sup>)</strong></td></tr><tr><td>Protein</td><td>73.63</td><td>112.52</td><td>56.95</td><td>61.03</td><td></td></tr><tr><td>Nucleotide</td><td>90.42</td><td>157.39</td><td>84.51</td><td>79.65</td><td></td></tr><tr><td>Ligand</td><td>73.44</td><td>116.06</td><td>61.34</td><td>67.07</td><td></td></tr><tr><td colspan="6"><strong>R.m.s. deviations</strong></td></tr><tr><td>Bond lengths (Å)</td><td>0.003</td><td>0.004</td><td>0.005</td><td>0.003</td><td></td></tr><tr><td>Bond angles (°)</td><td>0.636</td><td>0.678</td><td>0.779</td><td>0.640</td><td></td></tr><tr><td colspan="6"><strong>Validation</strong></td></tr><tr><td>MolProbity score</td><td>1.34</td><td>1.45</td><td>1.68</td><td>1.28</td><td></td></tr><tr><td>Clashscore</td><td>4.14</td><td>4.73</td><td>6.26</td><td>3.96</td><td></td></tr><tr><td>Poor rotamers (%)</td><td>0.02</td><td>0.00</td><td>0.00</td><td>0.00</td><td></td></tr><tr><td colspan="6"><strong>Ramachandran plot</strong></td></tr><tr><td>Favored (%)</td><td>97.23</td><td>96.68</td><td>95.13</td><td>97.55</td><td></td></tr><tr><td>Allowed (%)</td><td>2.77</td><td>3.30</td><td>4.83</td><td>2.45</td><td></td></tr><tr><td>Disallowed (%)</td><td>0.00</td><td>0.02</td><td>0.04</td><td>0.00</td><td></td></tr></tbody></table>
<table><caption>Table S2. Used oligonucleotides, Related to Key Resource Table.</caption><thead><tr><th>Name and usage</th><th>Source</th><th>Sequence (5' to 3')</th></tr></thead><tbody><tr><td>Template strand for scaffolds SC1 and SC2, used for RNA primer extension assays and cryo-EM sample prep (Pol III PTC)</td><td>This paper</td><td>CAAAATTTTCGGAAGGCATGC TCTGTGGCTTTGCTAAGAGAT TC</td></tr><tr><td>Scaffold 1 (SC1), used for RNA primer extension assays and cryo-EM sample prep (Pol III PTC), non- template strand</td><td>This paper</td><td>GAATCTCTTAGCAACCATTAT TTTTTTGCCCTTCCGAAAATTTT G</td></tr><tr><td>Scaffold 2 (SC2), used for RNA primer extension assays and cryo-EM sample prep (Pol III EC), non- template strand</td><td>This paper</td><td>GAATCTCTTAGCAACCATTAG CTTGCTGCCTTCCGAAAATTTT G</td></tr><tr><td>Template strand for promoter-dependent transcription assay, 5 dT-termination site</td><td>This paper</td><td>CAAAATTTTCGGAAGGCAAAA ATAATGGTTGCTAAGAGATTC GACGAACACATAGTTGCGAAA AAAACATTTTTTTATAGTAGCC GAAAATAG</td></tr><tr><td>Non-template strand for promoter-dependent transcription assay, 5 dT-termination site</td><td>This paper</td><td>CTATTTTCGGCTACTATAAAAA AATGTTTTTTTCGCAACTATGT GTTCGTCGAATCTCTTAGCAA CCATTATTTTTGCCTTCCGAAA ATTTTG</td></tr><tr><td>Template strand for promoter-dependent transcription assay, 4 dT-termination site</td><td>This paper</td><td>TCAAAATTTTCGGAAGGCAAA ATAATGGTTGCTAAGAGATTC GACGAACACATAGTTGCGAAA AAAACATTTTTTTATAGTAGCC GAAAATAG</td></tr><tr><td>Non-template strand for promoter-dependent transcription assay, 4 dT-termination site</td><td>This paper</td><td>CTATTTTCGGCTACTATAAAAA AATGTTTTTTTCGCAACTATGT GTTCGTCGAATCTCTTAGCAA CCATTATTTTTGCCTTCCGAAAA TTTTGA</td></tr><tr><td>Template strand for promoter-dependent transcription assay, 3 dT-termination site</td><td>This paper</td><td>TTCAAAATTTTCGGAAGGCAA ATAATGGTTGCTAAGAGATTC GACGAACACATAGTTGCGAAA AAAACATTTTTTTATAGTAGCC GAAAATAG</td></tr><tr><td>Non-template strand for promoter-dependent transcription assay, 3 dT-termination site</td><td>This paper</td><td>CTATTTTCGGCTACTATAAAAA AATGTTTTTTTCGCAACTATGT GTTCGTCGAATCTCTTAGCAA CCATTATTTTTGCCTTCCGAAAAT TTTGAA</td></tr><tr><td>Template strand for tailed-template transcription assay, 6 dT-termination site</td><td>This paper</td><td>GTTGGTAGTGGCGGTTGTAG GAAAAAAGATTGGAATTAGGT GGATGAGAGGGGTTATG</td></tr><tr><td>Non-template strand for tailed-template transcription assay, 6 dT-termination site</td><td>This paper</td><td>CCACCTAATTCCAATCTTTTTT CCTACAACCGCCACTACCAAC</td></tr><tr><td>Template strand for tailed-template transcription assay, 3 dT-termination site</td><td>This paper</td><td>GTTGGTAGTGGcGGTTGTAGG GTGAAAGATTGGAATTAGGTG GATGAGAGGGGTTATG</td></tr><tr><td>Non-template strand for tailed-template transcription assay, 3 dT-termination site</td><td>This paper</td><td>CCACCTAATTCCAATCTTTTCAC CCTACAACCgCCACTACCAAC</td></tr><tr><td>CRISPR-Cas9 donor, C128 Triple mutant, forward</td><td>This paper</td><td>CCAGACTATCTTATATTTCCGC ATTGGGTATGATGACAAGAAT TTCTTCTGCcTTTGAAGCtTCA GGtAAA</td></tr></tbody></table>
<table>
  <tr>
    <td>CRISPR-Cas9 donor, C128 Triple mutant, reverse</td>
    <td>This paper</td>
    <td>CAGTACAAAGCATACCAAATT GAGAAGGTTGTAATGCTCTTG GACCGGAAACTTTACCTGAAG CTTCAAAGG</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 Q199R mutant, forward</td>
    <td>This paper</td>
    <td>GTGGTTACTTTATTGTTAACG GTACCGAAAAAGTAATTCTTG TTCAAGAAagaTTATCaAAGAA TCGTAT</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 Q199R mutant, reverse</td>
    <td>This paper</td>
    <td>GGAGGCCTGAACAATGCCTTT CTTCTCATCTGCCTCCACAAT AATACGATTCTTtGATAAtctTTC T</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R451V mutant, forward</td>
    <td>This paper</td>
    <td>TGAACAGAGCTATCTCAACCG GTAATTGGTCTCTTAAAAGATT TAAGATGGAAGTTGCGGGTGT T</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R451V mutant, reverse</td>
    <td>This paper</td>
    <td>CATACCCAATGCGGAAATATA AGATAGTCTGGAAAGAACATG TGTAACACCCGCAACTTCCAT CT</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 K448A mutant, forward</td>
    <td>This paper</td>
    <td>GCGGACTGAACAGAGCTATCT CAACCGTAATTGGTCTCTTA AAAGATTTgctATGGAgAGGGC G</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 K448A mutant, reverse: 5'- -3'</td>
    <td>This paper</td>
    <td>CCCAATGCGGAAATATAAGAT AGTCTGGAAAGAACATGTGTA ACACCCGCCCTcTCCATagcAA ATC</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R481 mutant</td>
    <td>This paper</td>
    <td>TTATATTTCCGCATTGGGTATG ATGACAAGAATTTCTTCTCAGT TTGAAAAATCAGGTAAAGTTTC CGG</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R481 mutant</td>
    <td>This paper</td>
    <td>ATCTGCAGTACAAAGCATACC AAATTGAGAAGGTTGTAATGC TCTTGGACCGGAAACTTTACC TGATTT</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 H225 mutant, forward</td>
    <td>This paper</td>
    <td>GTGGAGGCAGATGAGAAGAA AGGCATTGTTCAGGCCTCCGT TACTTCATCTACCCTTGAAAGA AAAT</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 H225 mutant, reverse</td>
    <td>This paper</td>
    <td>TGCTTCAAATAGATCTTGCCAT TTTTGGTAATGACATAAGTCTT AGATTTTCTTTCAAGGGTAGAT</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R451-to-WT, forward</td>
    <td>This paper</td>
    <td>TGAACAGAGCTATCTCAACCG GTAATTGGTCTCTTAAAAGATT TAAGATGGAAAGAGCGGGTGT T</td>
  </tr>
  <tr>
    <td>CRISPR-Cas9 donor, C128 R451-to-WT mutant, reverse</td>
    <td>This paper</td>
    <td>CATACCCAATGCGGAAATATA AGATAGTCTGGAAAGAACATG TGTAACACCCGCTCTTTCCAT CT</td>
  </tr>
</table>
<figure><img src="image_51.png" /></figure>

Data S1. Data-collection and -processing workflow of the yeast Pol III PTC, Related to Figure 1.

(A) Cryo-EM data collection and processing strategy for the yeast Pol III PTC. Particle numbers are rounded down. Shown are the unsharpened cryo-EM maps obtain via 3D refinement in RELION. Reported resolution values are given below the cryo-EM maps and correspond to sharpened maps obtained via RELION post-processing. The resolution values of the unsharpened 3D maps are added in round brackets. Map features that show improved cryo-EM signal after masked 3D classification are highlighted in colours. Scale bar: 50 nm.

(B) Fourier shell correlation (FSC) curve of the yeast Pol III PTC derived via RELION post-processing (FSC = 0.143).

(C) Estimation of local resolution and angular distribution plot of the yeast Pol III PTC. Shown is the unsharpened cryo-EM map.
<figure><img src="image_52.png" /><figcaption>Data S2. Data-collection and -processing workflow of the yeast Pol III EC, Related to Figure 1.</figcaption></figure>

(A) Cryo-EM data collection and processing strategy for the yeast Pol III EC. Particle numbers are rounded down. Shown are the unsharpened cryo-EM maps obtain via 3D refinement in RELION. Reported resolution values are given below the cryo-EM maps and correspond to sharpened maps obtained via RELION post-processing. The resolution values of the unsharpened 3D maps are added in round brackets. Map features that show improved cryo-EM signal after masked 3D classification are highlighted in colours. Scale bar: 50 nm.

(B) Fourier shell correlation (FSC) curve of the yeast Pol III EC derived via RELION post-processing (FSC = 0.143).

(C) Estimation of local resolution and angular distribution plot of the yeast Pol III EC. Shown is the unsharpened cryo-EM map.
<figure><img src="image_53.png" /><figcaption>Data S3. Data-collection and -processing workflow of yeast Pol III Δ, Related to Figure 5.</figcaption></figure>

(A) Cryo-EM data collection and processing strategy for the yeast Pol III Δ. Particle numbers are rounded down. Shown are the unsharpened cryo-EM maps obtain via 3D refinement in RELION. Reported resolution values are given below the cryo-EM maps and correspond to sharpened maps obtained via RELION post-processing. The resolution values of the unsharpened 3D maps are added in round brackets. Scale bar: 50 nm.

(B) Fourier shell correlation (FSC) curve of the yeast Pol III Δ derived via RELION post-processing (FSC = 0.143).

(C) Estimation of local resolution and angular distribution plot of the yeast Pol III Δ. Shown is the unsharpened cryo-EM map.
<figure><img src="image_54.png" /><figcaption>Data S4. Data-collection and -processing workflow of yeast Pol III PTC + NTPs, Related to Figure 6.</figcaption></figure>

(A) Cryo-EM data collection and processing strategy for the yeast Pol III PTC + NTPs. Particle numbers are rounded down. Shown are the unsharpened cryo-EM maps obtain via 3D refinement in RELION. Reported resolution values are given below the cryo-EM maps and correspond to sharpened maps obtained via RELION post-processing. The resolution values of the unsharpened 3D maps are added in round brackets. Map features that show improved cryo-EM signal after masked 3D classification are highlighted in colours. Scale bar: 50 nm.
(B) Fourier shell correlation (FSC) curve of the yeast Pol III PTC + NTPs derived via RELION post-processing (FSC = 0.143).

(C) Fourier shell correlation (FSC) curve of the yeast Pol III PTC + NTPs map that was subjected to masked 3D-classification on the bound DNA/RNA (D/R) elements.

(D) Estimation of local resolution and angular distribution plot of the yeast Pol III PTC + NTPs. Shown is the unsharpened cryo-EM map.

(E) Estimation of local resolution and angular distribution plot of the yeast Pol III PTC + NTPs classified on the DNA/RNA elements. Shown is the unsharpened cryo-EM map.
<figure><img src="image_55.png" /></figure>

Data S6, Related to Figure 2, E. Multiple sequence alignment of *S. cerevisiae* C128 (Pol III) and its paralogs A135 (Pol I) and RBP1 Pol II. The sequence range is 190 to 490 in C128. Residues that form H-bonds with the NT-strand in the Pol III PTC are highlighted in red.