Article

The Biogenesis of SRP RNA Is Modulated by an RNA
Folding Intermediate Attained during Transcription
Graphical Abstract

Authors
Shingo Fukuda, Shannon Yan,
Yusuke Komi, Mingxuan Sun,
Ronen Gabizon, Carlos Bustamante

Correspondence
shingof0617@gmail.com (S.F.),
shannonyan@berkeley.edu (S.Y.),
carlosb@berkeley.edu (C.B.)

In Brief
SRP RNA begins to fold during its
synthesis by RNA polymerase. Fukuda
et al. find that SRP RNA exhibits a robust
co-transcriptional folding pattern,including (1) the initial formation of a nonnative obligatory intermediate thatenables RNA maturation and (2) its
eventual resolution into the native foldthat confers SRP functionality.

Highlightsd

SRP RNA exhibits robust co-transcriptional folding invariant
to transcription ratesd

SRP RNA adopts a non-native obligatory intermediate prior toits functional foldd

The obligatory intermediate enables SRP RNA maturation
during early transcriptiond

Altering the non-native-to-native transition in SRP RNA
impacts cell viability

Fukuda et al., 2020, Molecular Cell 77, 1–10
January 16, 2020 Published by Elsevier Inc.
https://doi.org/10.1016/j.molcel.2019.10.006

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Molecular Cell

ArticleThe Biogenesis of SRP RNA Is
Modulated by an RNA Folding
Intermediate Attained during Transcription
Shingo Fukuda,1,2 ,3 ,12 ,* Shannon Yan,1,4 ,12 ,* Yusuke Komi,1,5 Mingxuan Sun,1,4 Ronen Gabizon,1and Carlos Bustamante1,4 ,6 ,7 ,8 ,9 ,10 ,11,13 ,*1Institute for Quantitative Biosciences-QB3, University of California, Berkeley, Berkeley, CA, USA
2Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Science, Tokyo, Japan
3Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan
4Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA
5Laboratory for Protein Conformation Diseases, RIKEN Center for Brain Science, Wako, Saitama, Japan
6Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, CA, USA
7Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, USA
8Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA
9Department of Physics, University of California, Berkeley, Berkeley, CA, USA
10Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA
11Kavli Energy Nanoscience Institute, University of California, Berkeley, Berkeley, CA, USA
12These authors contributed equally
13Lead Contact*Correspondence: shingof0617@gmail.com (S.F.), shannonyan@berkeley.edu (S.Y.), carlosb@berkeley.edu (C.B.)
https://doi.org/10.1016/j.molcel.2019.10.006

SUMMARY

The signal recognition particle (SRP), responsible forco-translational protein targeting and delivery tocellular membranes, depends on the native longhairpin fold of its RNA to confer functionality. Since
RNA initiates folding during its synthesis, we usedhigh-resolution optical tweezers to follow in realtime the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription
rate changes and the presence or absence of its
5 0 -precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native
hairpin intermediate (H1) that eventually rearranges
into the native fold. Furthermore, H1 provides a
structural platform alternative to the native fold for
RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are
detrimental to the cell, altogether showing that a
co-transcriptional folding pathway underpins theproper biogenesis of function-essential SRP RNA.

INTRODUCTION
Nascent RNA transcripts, like their protein counterparts, begin to
fold during their synthesis, before the entire sequence iscompleted and released from RNA polymerase (RNAP) (Al-Hashimi and Walter, 2008; Pan and Sosnick, 2006; Perales andBentley, 2009). This scenario suggests that the co-transcriptional folding landscape of RNA may differ from that of full-length

RNA and possibly contain alternative folding intermediates. The
set of accessible intermediates may be particularly significant forfunctional RNAs, whose essential cellular activity hinges tightly
on the attainment of specific folds. The mechanism underlying
co-transcriptional RNA folding has been investigated intensely
by means of biochemical bulk assays as well as computational
methods (Chao et al., 1995; Heilman-Miller and Woodson,
2003; Lewicki et al., 1993; Nechooshtan et al., 2014; Palangat
et al., 1998; Pan et al., 1999; Wong et al., 2005, 2007; Zhang
and Landick, 2016; Zhao et al., 2011). Single-molecule force
spectroscopy was also employed to monitor the folding of apbuE adenine riboswitch and its conformational switching
upon ligand binding (Frieda and Block, 2012). However, the rolesof the co-transcriptional intermediates in the functionality ofessential RNAs and the viability of the cell have not been addressed in depth.
The RNA core of the signal recognition particle (SRP), a highly
conserved protein biogenesis machinery, is a suitable model
system to study the co-transcriptional folding of RNA (Doudna
and Batey, 2004; Keenan et al., 2001). This 114-nt SRP RNA
from Escherichia coli is known to fold into a simple, !50-bplong functional hairpin. It is in this native fold that SRP RNA
orchestrates elaborate and critical conformational rearrangements of the SRP proteins (Ffh and FtsY), which activate SRP
for co-translational protein delivery to the appropriate translocation machineries in cell membranes (Figure 1A) (Akopian et al.,2013). Specifically, SRP RNA recruits Ffh with pico-molar affinity
to its hairpin tip-proximal stem (next to the hairpin tetra-loop); thecomplex then targets ribosome cargos displaying specificnascent polypeptide chains. Next, the G-domain of Ffh (G for
GTPase) associates with the G-domain of the membrane-bound
SRP receptor protein FtsY, thereby bringing cargos into the vicinity of translocons. To initiate cargo unloading, the G-domains

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Figure1. Protein-Targeting
Activity
Orchestrated by the Native Long-Hairpin
Fold of SRP RNA(A) Left to right: the signal recognition particle(SRP) in E. coli is composed of SRP RNA (red) andan SRP protein, Ffh (blue), whose M-domain binds
to RNA hairpin-tip proximal stem. This ribonucleoprotein complex recognizes nascent signaling
peptides (purple) displayed on the ribosome cargos (gray), which are then brought to the translocons (brown barrels). The SRP:cargo complex
first interacts with the membrane-bound SRP receptor protein, FtsY (green), via the G-domains ofFfh and FtsY. Next, the two G-domains swing fromthe SRP RNA hairpin tip (black arrow) and dockonto the hairpin-tip distal stem. This conformational rearrangement is the crucial checkpoint thattriggers GTP hydrolysis on the two proteins (purple
dot for GTP; orange for GDP) and activates cargo
unloading to the translocon, through which thesynthesized polypeptide is transported.
(B) The native, functional long-hairpin fold of 114nt-long SRP RNA from E. coli. The hairpin-tip
proximal stem where Ffh M-domain binds ishighlighted in blue, and the hairpin tetra-loop region where the SRP-FtsY assembly initially interacts is in green. The hairpin-tip distal stem highlighted in purple is the G-domain docking site, which is essential for SRP protein-targeting activity. Blunt-ended
truncations (colored dash lines) on the long-hairpin fold of SRP RNA incrementally lower SRP GTPase activity and eventually impair SRP-protein-targeting
functionality (black, no impact; purple, lowered GTPase activity; red, loss of activity).

must rearrange their conformation, swinging together from thehairpin tetra-loop end to dock on the hairpin tip-distal stem,
which comprises nucleotides 10–15 and 96–101 of the SRP
RNA (Heilman-Miller and Woodson, 2003; Jagath et al., 2001;
Peterson and Phillips, 2008; Shen et al., 2013; Siu et al., 2007).
This docking configuration subsequently activates guanosine
triphosphate (GTP) hydrolysis and signals cargo unloading.
When the distal stem of SRP RNA is shortened by more than
14 bp (Figure 1B), the docking configuration is impaired, which
attenuates GTPase activity and abolishes ribosome cargo transport (Ataide et al., 2011). Therefore, the attainment of the SRPRNA native, long-hairpin fold is essential for SRP functionality.
Here we use high-resolution optical tweezers (Comstock et al.,2011; Gabizon et al., 2018; Righini et al., 2018) to follow in realtime the co-transcriptional folding of 4.5S RNA, the prematureform of SRP RNA—as naturally transcribed by RNAP from theffs gene—with a 24-nt-long precursor sequence appended atits 50 end, and compare it to that of mature SRP RNA (Bothwell
et al., 1976; Hsu et al., 1984). We find that SRP RNA folding requires a partially folded intermediate that must later be resolved
into the correct state. This intermediate raises many questions
about co-transcriptional folding: does the vectorial (50 to 30 ) nature of RNA synthesis favor the folding and sequestering of the
upstream RNA sequences into non-native intermediate structures? Are on-pathway folding intermediates always productive(i.e., part of the final native state), or, alternatively, can a nonproductive intermediate be on pathway (i.e., obligatory) co-transcriptionally? How does the rate at which the nascent RNA
emerges from RNAP alter the folding process? Does RNAP
pausing (Artsimovitch and Landick, 2000; Gabizon et al., 2018;
Larson et al., 2014) play a role in determining the folding

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pathway? As a functional RNA transcript emerges from RNAP,
do changes in the population and residence time of the moleculein intermediate states lead to any biological consequences?
Hence, we also characterize in vivo the effect from mutant SRP
RNAs with altered folding dynamics to interrogate the physiological relevance of co-transcriptional RNA folding.
RESULTSSRP RNA Co-transcriptional Folding Is Robust
To observe how SRP RNA naturally attains its 50-bp-long hairpinfunctional fold, we use a high-resolution dual-trap optical tweezers to tether a single-polymerase-RNA nascent chain complex
and monitor the tether end-to-end extension as the RNA
emerges and folds on the surface of E. coli RNAP. Specifically,
the RNAP-RNA complex is tethered between DNA handles andconnected to a pair of surface-modified polystyrene beads
manipulated by infrared (IR) laser traps (Figure 2A; STAR
Methods). The nascent complex is stalled after the T7A1 promoter at a short G-less sequence located before the ffs gene(pre-SRP RNA, 24 + 114 nt) or before the mature, shortened
version (114 nt; STAR Methods) and held under a low constanttension (8.6 pN) to permit the folding of nascent transcript.
Nucleotide triphosphates (NTPs) at various concentrations(10 mM to 1 mM; KM !250 mM) (Harada et al., 2001) were thendelivered to restart transcription. A typical co-transcriptional
folding trajectory of extension versus time is shown in Figure 2B(y axis, extension in nanometers; x axis, time in seconds). The
positive slope in the trajectory reflects the continuous growthof RNA (G in Figure 2B), while a sudden decrease (i.e., verticaldrop) in tether extension reflects a folding transition (F). The

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Figure 2. Robust Co-transcriptional Folding of SRP RNA Resolved by Single-Molecule Optical Tweezers(A) An E. coli RNA polymerase (RNAP) elongation complex is tethered via a pair of double-stranded DNA handles and held on the optical tweezers. Active
transcription at different [NTP] is monitored in real time, where the growth and folding of nascent RNA is detected as the tether extension change under constantforce (STAR Methods).
(B) A representative co-transcriptional folding trajectory of SRP RNA recorded at 8.6 pN and 50 mM NTP. The positive slope in tether extension reflects the gradual
growth (G) of nascent transcript; the horizontal segments (i.e., no change in extension over time) signify transcriptional pauses (P1 and P2). A decrease inextension indicates RNA folding (F), which is seen as a vertical drop in the trajectory, or a negative slope if the transcript growth and the folding occur concurrently(G + F). Hopping dynamics in the trajectory (region highlighted in red) reveal that the transcript rapidly unfolds and refolds, manifesting transitions between
competing structural folds. After completing the transcription of SRP RNA encoded in the template DNA (black arrow), RNAP continues to transcript a short DNA
spacer (region colored in black) before it runs off the template DNA and breaks the tether (see STAR Methods). This last stretch of monotonic/feature-less RNA
extension growth serves as an ending landmark for a valid trajectory recording. See main text for assignments on intermediate folds H1, H2, and H3.
(C and D) Representative co-transcriptional folding trajectories of pre-SRP RNA (24 + 114 nt) (C) and mature SRP RNA (114 nt) (D) at different transcription rates(see timescale bar) as a function of [NTP] (n = 41, 37; F = 8.6 pN).
(E) A schematic cartoon illustrates the folding evolvement of nascent SRP RNA as it is transcribed by RNAP.

horizontal segments (i.e., tether length invariant over time) correspond to transcriptional pauses of RNAP on the template DNA(P), whereas regions of gradual negative slope in the trajectory
are periods where RNA growth and folding occur concurrently(G + F) with the nascent transcript. That is, as one nucleotide
newly emerges from the RNAP, it readily base pairs/condenses
with another complementary nucleotide in the upstream sectionof the RNA nascent chain, thus leading to a shortening of thetranscript extension by a total of two nucleotides.
Surprisingly, the co-transcriptional folding trajectory of SRP
RNA is invariant and remains robust across three orders ofmagnitude change in NTP concentration (Figure 2C for three examples of pre-SRP RNA, 24 + 114 nt, n = 41; Figure 2D for
mature SRP RNA, 114 nt, n = 37; also Figure S1A). Indeed, trajectories with vastly different transcription rates, all within the physiological range (i.e., from !1 nt/s at 10 mM NTP to !50 nt/s at
1 mM NTP), can be rescaled by their total duration to super-

impose onto one another. This observation is contrary to cases
wherein RNA-folding trajectories are drastically altered by heterologous polymerases from bacterial phages synthesizing E. coli
RNAs at artificially high rates and with different transcription
pausing patterns (Lewicki et al., 1993). Furthermore, Figures
2C and 2D show that the folding trajectories obtained for the precursor-bearing and the mature RNA sequences are identical,
other than the fact that the former trajectory is simply appended
with an extra extension feature illustrating the initial synthesis of
24-nt-long precursor fragment (highlighted in magenta). Hence,in the remainder of this study, except for places noted explicitly,
we use the mature SRP RNA template (114 nt).
An Obligatory Non-native Intermediate
We find that the folding trajectories of SRP RNA consistently andreproducibly comprise three main features (Figure 2B): (1) a small
hump (highlighted in dark blue); (2) two long plateaus (highlighted

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Figure 3. Modulate Co-transcriptional Folding Intermediates by Antisense Oligos and Point Mutations(A) Structures of SRP RNA co-transcriptional folding intermediates: H1, H2, H3, and H4 hairpins. Antisense oligo-1 and oligo-2 respectively target the 50 arms of
H1 and H2. U18C mutation stabilizes H1, and U32C/U34C stabilizes H3. The self-complementary region on SRP RNA 50 stretch sequesters the transcript 50 end in
H1 fold; the self-complementary 30 stretch of SRP RNA, eventually base-pairing with the 50 stretch to form the hairpin-tip distal stem, similarly sequesters thetranscript 30 end in H4.
(B and C) Representative co-transcriptional folding trajectories of wild-type SRP RNA recorded with 10 mM antisense oligo-1 (dark red) (B) and oligo-2 (orange; at
50 mM NTP) (C); the wild-type trajectory without antisense oligos (black) is shown for comparison. Oligo-1 eliminates the initial drop in extension (dark red arrows)that signifies H1 formation (G + F; highlighted in dark yellow) and the subsequent hopping dynamics (dashed-line boxes) that indicate structural rearrangements of
H1 into the native fold. Oligo-2 prevents H2 folding (i.e., the hairpin tip-proximal stem in yellow; Figure 2E), thereby generating an upward-going trace absent of the
middle plateau feature (orange and black arrows; gap between gray dashed lines).
(D–F) Representative co-transcriptional folding trajectories of U32C/U34C (red) (D), wild-type (black) (E), and U18C (cyan) (F) transcripts (F = 8.6 pN; 50 mM NTP).
Hopping dynamics near the end of transcription (red box region in E and F) are attenuated with the U18C mutation, signifying that the structural transition toward
the long-hairpin native fold becomes less favored. Instead, a hump-like feature is detected (red box zoom-in in F; similar to that of H1 formation, cyan arrows),
indicating that the transcript 30 stretch samples a small hairpin fold (H4 in A) before attaining the native fold.
(G and H) Histograms on the magnitude of hopping dynamics show that attempts to transition from H1 to the native fold is attenuated with the U18C mutant (cyan;n = 99) (H) as compared to the wild type (gray; n = 153) (G).

in light blue and yellow, respectively), where the latter one isembedded with spikes; and (3) a distinct hopping feature in
RNA extension (highlighted in red) occurring just before thecompletion of SRP RNA synthesis (Figure 2B, black arrow). To
assign the folding features in the trajectory to specific portionsof the nascent RNA (see sections below), we performed additional non-equilibrium co-transcriptional folding experiments under three conditions: (1) in the presence of antisense oligos toblock particular secondary structural folds (Figures 3B and 3C),
(2) with transcripts harboring mutations to promote and stabilize
those folds (Figures 3D–3F), and (3) under depleting NTP conditions to lengthen specific transcriptional pauses (Figures S1B
and S1C). We also collected unfolding force-extension (F-X)
curves to examine intermediate folds adopted by nascent transcripts of incremental length stalled on RNAP at equilibrium (Figures S2A, S2B, S2D, and S2E; STAR Methods) and RNA extension hopping experiments to interrogate folding transitions
between specific intermediates at quasi-equilibrium (Figure S3;STAR Methods).
The first hump in the trajectory corresponds to the initial
growth of the nascent transcript until it reaches a length of
17–20 nt, followed by a subsequent folding transition in which

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it forms a small intermediate hairpin, H1 not present in the native
fold (Figure 2E, 2 and 8). Note that after this initial folding event,the RNA extension drops approximately to the same level as thestarting point of the trajectory (Figure 2B). This initial hump for
non-native-like H1 folding is present at all transcription rates
tested (Figures 2C and 2D) and hence co-transcriptionally obligatory but becomes an upward slope in the presence of antisense
oligo-1 (dark red traces in Figure 3B), which is complementary to
the first 10 nt of SRP RNA (Figure 3A). In this case, the H1 folding
is now outcompeted by the antisense oligo-1, and the RNA
tether extension becomes larger (Figure 3B, difference between
gray dashed lines), because the right-arm stretch of H1 remains
as a single strand.
After the first intermediate hairpin (H1) forms, RNAP pauses(P1, light blue in Figure 2B), then resumes transcription foranother !55 nt (green in Figure 2B) and pauses again (P2, yellow
in Figure 2B). Both pause durations increase when [UTP] (concentration, in molar, of uridine 5’-triphosphate) is selectively
decreased during transcription, while the second pause, P2,also lengthens at low [GTP] (Figure S1C). To identify which
U or G in the transcript sequence corresponds to P1 and P2,
we performed the same real-time RNA synthesis experiments

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

under high tension (!22 pN) to increase the spatial resolution of
the transcription trajectory and to prevent folding transitions.
(Righini et al., 2018) We found that P1 occurs at U36 (36.2 ± 2.5
nt into transcription, indexed as the mature SRP RNA sequence;
Figure S4), whereas P2 occurs around U82/U84—a known prominent pause site that has been shown to be suppressed by basepairing compensating mutations U82A/A25U.(Wong et al., 2007)
In the presence of these mutations, P2 shortens significantly,
confirming our assignment. The G73U82G83 stretch around P2
matches the consensus pause element G-10Y"1G+1 describedfor RNAP (Vvedenskaya et al., 2014) and resulting in the strong
G-pause detected at P2. As for P1, its U-rich sequence stretch
U34G35U36U37U38, and similarly the U82G83U84 stretch at P2,
resemble another pause-promoting element (U"2G"1) reported
previously for E. coli RNAP (Hein et al., 2011), hence resulting
in long transcriptional pauses under limiting [UTP]. Therefore,
the occurrences of P1 and P2 are independent of RNA secondary structure formation, consistent with their persistence in thefolding-free high-tension trajectories. We also found that these
pauses do not involve backtracking of RNAP, since they areinvariant to the addition of GreB, a transcription factor that rescues RNAP from backtracking pauses (Figures S1E and S1F)
(Toulmé et al., 2000).
RNA-Fold Sampling and Transition to Native State
During P2, the emerged nascent transcript is !70 nt long andspends most of the time in a partially folded state that contains
the non-native intermediate hairpin H1 on the 50 end and a second hairpin H2 !24 nt downstream (➃ in Figure 2E). Interestingly,
at this point we detect upward and downward spikes of discrete
sizes in RNA extension (magenta arrowheads in Figure 2B),
which are different structural folds accessible and transiently
visited by the newly synthesized stretch of SRP RNA (Figure 2E,
yellow segment in 3). This behavior corresponds to hairpin H2
unfolding (i.e., the large extension, upward spikes observed on
P2 and quickly refolding into a more compact but short-lived
state, hairpin H3 (➃0 in Figure 2E; the downward spikes). As
before, to confirm our assignment, we specifically inhibited H2folding with an antisense oligo-2 (Figure 3A), which renders theright-arm stretch of H2 unpaired and leads to an upward shift
in extension for the P2 region of the trajectory (Figure 3C, difference between gray dashed lines). Thus, H2 constitutes thehairpin tip-proximal stem of the native fold, known to provide
the essential binding site for protein Ffh to associate with SRP
RNA (Figure 1B, blue-shaded region). To confirm the assignmentof hairpin H3, we use specific point mutations, U32C/U34C, thatshould preferentially stabilize this putative state. The co-transcriptional folding trajectory recorded with these mutants demonstrates a clear lengthening of residence time in the H3 state;i.e., the downward spikes in P2 become well-resolved dwells
as seen in Figure 3D (red traces).
Eventually, RNAP exits pause P2 and resumes transcription,
adding nucleotides to the 30 end of the !70-nt-long nascent
SRP RNA. In this part of the folding trajectory, however, theRNA extension exhibits a negative slope (purple region in Figure 2B), whose magnitude is [NTP] dependent, with lower[NTP] leading to shallower slopes (Figure S1A). This observation
indicates that in this region, RNA growth and folding occur

concurrently, wherein the newly synthesized section of RNA(!31 nt) readily base pairs with the upstream complementary
single-stranded region located between the two intermediate
hairpins, H1 and H2 (Figure 2E, ➄; purple zipping with green).
At this point, !60% of the final long-hairpin native fold of SRP
RNA is completed, while !20 nt at its 50 end are sequesteredin the non-native intermediate hairpin fold H1.
As the SRP RNA continues to grow (from !101 nt to 114 nt;
Figure 2B, region in red), we observed distinct hopping in extension, reflecting the rapid folding (low extension) and unfolding(large extension) of the nearly completed transcript as it samples
competing folding states. The histogram of hopping amplitude
shows an average of 4.5 ± 1.5 nm and maximum at !8 nm (Figure 3G), which corresponds to an extension gain of !21 nt (under
8.6 pN of tension), suggesting transient multiple attempts at unfolding and resolving non-native hairpin H1. This interpretation iscorroborated by the aforementioned antisense oligo-1 control
experiment; when the initial formation of H1 is abolished, thehopping dynamics are eliminated near the end of transcription(black-dashed boxes in Figure 3B). These correlated featuresin the trajectory indicate that the hopping dynamics reflect astructural bi-stability during which the !14-nt RNA 30 end gradually shifts the folding equilibrium away from the state in which
the RNA 50 stretch is organized into hairpin H1 to a state in which
the 50 stretch is released to anneal with the emerging complementary 30 stretch (Figure 2E, ➅–➆). The negative slope preceded by the hopping feature evidences the occurrence ofsuch a concurrent RNA growth and folding (Figure 2B, region
in red), which leads to the final native state of SRP RNA. To
examine the structure attained by this completed SRP RNA,
we ramped up the tension to unfold the RNA at the end of theco-transcriptional trajectory; we observed a single unfolding
step of !114 nt in extension gain (Figure S2C). This value
matches the number of nucleotides involved in the SRP RNA
native fold; the single cooperative unfolding transition again
supports the attainment of the native long hairpin state, whichcontains the hairpin tip distal-stem essential for SRP’s proteintargeting activity.
Co-transcriptional Maturation of SRP RNA
The co-transcriptional folding trajectory of SRP RNA, besides
being robust to vastly different transcription rates, is also
invariant to the presence of 24-nt 50 -precursor sequence. We
hence ask whether, given its inert impact on folding, this 50 precursor might be cleaved off sometime during transcription. In thecell, the 50 precursor must be removed, either during or after
transcription, in order to generate the 114-nt mature SRP RNA.
While the field generally assumes that maturation of functional
RNAs occurs after the transcripts are synthesized and released
from RNAP, the exact timing of 50 cleavage is unknown. Yet, existing studies on co-transcriptional folding of SRP RNA havebeen conducted mostly with the mature sequence lacking the
50 precursor (Wong et al., 2007). The fact that the 138-nt preSRP RNA is not isolated from bacteria unless the processing
enzyme RNase P, which is five times less abundant, is impaired,
which generates a mixture of pre- and mature products, suggests an extremely rapid or co-transcriptional maturation of
SRP RNA (Altman and Kirsebom, 1999; Bothwell et al., 1976;

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Figure 4. Obligatory Intermediate H1 Enables Maturation of SRP
RNA during Early Transcription(A) Nascent pre-SRP RNA long enough to form hairpin H1 (purple) comprises
the 24-nt 50 precursor (magenta), the 22-nt mature sequence (dark blue), andanother 18 nt (green) covered by RNAP (gray). This intermediate possesses a
50 -U"1G+1/C21R22-30 base-pair sequence at its short-stem hairpin junction (redarrow), similar to the cleavage site found in the native long-hairpin, and hence
may be processed by E. coli RNase P. Four RNA:RNAP stalled complexes withincreasing transcript length, 24 + 22 (+18), 24 + 30 (+18), 24 + 66 (+18), 24 + 114(+18), and full-length released transcript, 24 + 114D, as control, are treatedwith RNase P to examine their propensity of co-transcriptional maturation(STAR Methods).
(B) Urea PAGE gel (7 M, 12%) resolves RNA fragments cleaved from the stalled
RNAP complexes upon RNase P treatment (n = 5 replicates; negative controls
in the left gel). Both the early stalled 24 + 22 (+18) complex and the full-length
species, 24 + 114 (+18) and 24 + 114D, show a !24-nt precursor band indicating RNase P processing. In this gel range, the !40-nt-long 30 side productcleaved from the 24 + 22 (+18) transcript is also detected.
(C) Urea PAGE gel (7 M, 12%) resolves cDNAs reverse transcribed from theabove RNA samples using a common DNA primer (n = 5 replicates; negative
controls in the left gel). Upon RNase P treatment, both the 24 + 22 (+18)
complex and the full-length species, 24 + 114 (+18) and 24 + 114D, show twobands, a !64-nt cDNA from the un-processed transcript and a !40-nt cDNAfrom the cleaved 30 -side RNA, which is sequenced to verify the cleavage site at
the 50 -U"1G+1 (STAR Methods).

Dong et al., 1996) Therefore, we looked into each intermediate
detected along the SRP RNA folding trajectory for specific structural and sequence elements that match those recognized by
RNase P, namely, a hairpin stem at least !3–4 bp in size with little
to no 30 end single strand and a 50 -U"1G+1/C21R22-30 base-pairing sequence at its hairpin junction (Figure 4A) (Li et al., 2005;
McKinney et al., 2001; Peck-Miller and Altman, 1991; Sinapah

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et al., 2011). RNase P reportedly cuts between the U"1 andG+1 to remove the 50 precursor and yields mature functional
RNA substrates, including tRNAs (McKinney et al., 2001; Sinapah et al., 2011). We found that both the final native long-hairpin
fold and the nascent obligatory non-native intermediate hairpin
H1 possess the above features required for RNase P processing,and the latter may allow the possibility of co-transcriptional
maturation.
To examine if RNase P indeed recognizes intermediate hairpin
H1 and hence cleaves off the 24-nt 50 precursor from the nascent
SRP RNA during transcription, we prepared—in the presence/
absence of RNase P—four stalled RNAP-RNA complexes displaying transcripts of various length and compared how their
RNA substrates are processed by the enzyme (STAR Methods).
Specifically, these four complexes correspond to evolving time
points along the folding trajectory: around the time when H1 first
appears (24 + 22 nt and 24 + 30 nt; Figure 2E, ➁–➂) and frommidway (24 + 66 nt; Figure 2E, ➃) to complete transcription(24 + 114 nt; Figure 2E, ➇). We find that a small !24-nt RNA fragment appears only when the 24 + 22-nt and 24 + 114-nt RNAPRNA complexes are prepared in the presence of RNase P,
whereas the 24 + 30-nt and 24 + 66-nt complexes show no signsof such fragments (Figure 4B). We next use a common primer,
fluorophore labeled, to reverse transcribe the RNAs from thesecomplexes. Consistently, in the presence of RNase P, the 24 +30-nt and 24 + 66-nt complexes show only one band representing cDNAs of uncut transcripts, while both the 24 + 22-nt and24 + 114-nt complexes show one additional lower band, which
indeed is !24 nt shorter, reflecting the 50 truncation on the processed transcripts (Figure 4C). Judging from the fluorescence intensity ratio between the two cDNA bands, !10% of the transcripts from the 24 + 22-nt complex are processed compared
to !90% processed from the 24 + 114-nt complex (Figure 4C;
the 24 + 114-nt transcripts released from RNAP, as a control,
are also processed to a similar level). Sequencing of the lower
band cDNA reveals the initial bases on its RNA template 50 end
and confirms the RNase P cleavage site located between the
aforementioned U"1G+1 section. These results hence demonstrate two time points at which maturation of SRP RNA is
feasible: (1) during early transcription, when the nascent RNA
is just long enough to form hairpin H1; and (2) when the entire
SRP RNA has emerged and is available to attain the native
long-hairpin fold (which is the default substrate in traditional
RNase P characterization) (Peck-Miller and Altman, 1991). We
speculate that the RNAP pause at the P1 site, right after H1 formation, may grant an adequate, seconds-long window for
RNase P to bind H1 and process the nascent transcript cotranscriptionally.
Altered Folding Dynamics Impact Viability
The intermediate hairpin H1 on the nascent SRP RNA 50 end, as
shown above, enables the timely maturation of the RNA duringinitial transcription. Nevertheless, this non-active intermediate
must also be resolved timely, as reflected in the hopping dynamics toward the end of folding trajectories, so that the RNA
50 stretch sequestered by H1 can anneal with the emerging 30
stretch to attain the activity-essential distal hairpin stem present
in the SRP RNA native fold (Figure 1B). Accordingly, we wonder

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Figure 5. Intermediate-Stabilizing Variants of SRP RNA Impact Cell Viability(A and B) Cell viability assay of E. coli transformed with plasmids carrying SRP RNA variants, which shift the folding equilibrium away or delay the folding kinetics
toward the native fold (n = 6 replicates; STAR Methods).
(A) Bacteria plate: transcription of full-length wild-type SRP RNA (WT_114) shows normal growth as that of the control conditions (empty plasmid and transcript
read-through), whereas the 30 -truncation variants (SRP_100, U18C_100, and U32C/U34C_100) are lethal. Surviving colonies were sequenced and identified toharbor modified plasmids, which prevents transcription of specific SRP RNA variants, thereby alleviating the lethal impact (Figure S5B).
(B) Bacteria plate: for full-length SRP RNA variants containing point mutations to stabilize specific co-transcriptional folding intermediates, U32C/U34C_114
shows growth similar to that of WT_114, while U18C_114 exhibits a reduction in surviving colonies. Relative colony counts: WT_114, 100%; U32C/U34C_114,!87%; and U18C_114, !44%.
(C and D) Differences in toxicity of full-length SRP RNA mutants were resolved by their cell growth profiles post-selective (C) and post-permissive (D) growth
condition (i.e., +Kan/+Kan and "Kan/+Kan; Kan, kanamycin; n = 5; STAR Methods). U18C_114 causes clear lengthening of the lag phase.
(E and F) Comparisons of selective growth profiles in (C) and (D). (E) Boxplot: growth rate in log phase; (F) Boxplot: halfway time reaching the stationary phase as aproxy of lag phase duration (p values calculated from two-sample t test; red mark represents the median; blue box shows the 25th–75th percentile; whisker shows
maximum and minimum values; n.s., not statistically significant).
(G) Summary on the cell viability and toxicity impact of SRP RNA variants examined in this work.

what the biological consequences are when this function-criticalstructural rearrangement of SRP RNA is perturbed. Hence, wesought to introduce mutations in SRP RNA, either to slow
down the RNA-folding kinetics or shift the equilibrium between
the intermediate and the final native fold, and characterize theireffect in vivo (Figure 5).
To delay the structural transition toward the native fold without
altering the final equilibrium, we employ point mutations tolengthen the lifetime of specific co-transcriptional intermediates,
such as the obligatory H1 hairpin stabilized by U18C (U18C_114;
Figure 3F) and intermediate H3 by U32C/U34C (U32C/
U34C_114; Figure 3D). The final folding state of these point
mutant full-length transcripts has been verified to be identicalas that of the wild-type full-length SRP RNA (WT_114), where

they readily acquire in vitro the same native long-hairpin foldthat exhibits comparable stability as that of the wild-type transcript and unwinds cooperatively in a single step (Figures S2Gand S2H).
To shift the equilibrium away from the native fold, on the other
hand, we use 30 -truncated mutants (WT_100, U18C_100, U32C/
U34C_100), because according to the folding trajectories, theincomplete !100-nt transcript rapidly transitions between the
H1-bearing and native-like folds and attains comparable populations (40% and 60%; Figure S3C). Note that this native-like fold(i.e., the partial-native fold [PN] in Figure S3C) acquires theminimal G-domain docking site in the hairpin-tip distal stem (purple-highlighted region in Figure 1B), which is essential for SRP
activity. Combining truncation and specific point mutations

Molecular Cell 77, 1–10, January 16, 2020 7

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

allows us to further bias the folding equilibrium toward theH1-bearing form (e.g., 89.8% and 10.2% with U18C_100;
Figure S2H).
Preparation of plasmids harboring these potentially toxic SRP
RNA variants is done with a special E. coli strain, which tightly
represses transcription of genes governed under specific promoter/operator elements (see STAR Methods; Figure S5A).(Lutz
and Bujard, 1997) Next, a standard BL21 strain (since knockoutof SRP RNA is inviable) is transformed with these plasmids tooverexpress specific SRP RNA variants, while the endogenous
4.5S RNA is still transcribed by E. coli polymerase and processed by RNase P. It is known that the endogenous SRP
RNA is in four times excess of the SRP protein Ffh (Jensen
et al., 1994) and that the exogenous transcripts outnumber theendogenous ones. Hence, in the in vivo experiments, we expectthat the majority of Ffh to be bound/sequestered by the SRP
RNA variants (i.e., associated with the hairpin tip-proximal
stem shown in Figure 1B). Accordingly, for different transcript
variants, their ability to rearrange non-native intermediates(e.g., resolving hairpin H1 hairpin) into the native long-hairpin
fold determines the competency of SRP complex and ultimately
the cell viability.
As shown in Figure 5A, synthesis of exogenous full-length
wild-type SRP RNA (WT_114) has no impact on cell viability,
yielding normal growth similar to that seen with the empty
plasmid control. In contrast, transcription of truncated SRP
RNAs is lethal to E. coli, with few colonies surviving (bottom
half: WT_100, U18C_100, U32C/U34C_100). We sequenced
the few colonies surviving in the SRP RNA variant experiments
and found that these bacteria harbored plasmids with either mutations or truncations in the promoter or terminator (Figure S5B),
which prevent the precise synthesis of the intended transcripts
and likely alleviate the lethal impact of those plasmids. Indeed,
the terminator-omitting/read-through plasmid control (Figure 5A), though inserted with a truncated variant (WT_100),
showed no deterioration of viability. Interestingly, it has been reported that when the SRP RNA hairpin-distal stem are truncatedby !10-bp (black-dashed line in Figure 1B)—namely, shortening
both the 50 and 30 ends—SRP activity remains unaffected.(Shenet al., 2013) In other words, removing the last 14 nt on the SRPRNA 30 end does not take away any of the nucleotides thatform the essential G-domain docking site in the native longhairpin fold. Therefore, it is the intact 50 stretch present in theasymmetrically truncated 100-nt-long transcripts that tips thefolding equilibrium of SRP RNA away from the native fold (e.g.,
from 100% population to 60% with the wild-type sequence)and is sufficient to render a lethal phenotype.
As for the SRP RNA full-length variants that alter folding kinetics, while U32C/U34C appears indistinguishable to that ofwild type in the cell viability plate assay, U18C exhibits a moderate reduction in surviving colony counts (Figure 5B); thisdecline is aggravated under stress condition (e.g., high-salt
growth medium; Figure S5C). Differences in toxicity of thesepoint mutants are then evaluated by their plasmid loss in transformed cells under selective versus antibiotic-free (permissive)
growth conditions and their subsequent recovery growth profiles under selection (STAR Methods) (Chen et al., 2017). We
find that both point mutants, compared to wild type, exhibit a8

Molecular Cell 77, 1–10, January 16, 2020

significant lengthening in the lag phase during growth (Figure 5F), indicating their impact in delaying cell maturation,
which requires efficient protein production as well as protein
delivery by SRP complexes before cells can divide and multiply
in the log phase. Moreover, consistent with the above viability
assay, U18C is the more toxic variant, because it also causesa slower growth rate in log phase (Figure 5E) and a tendency
to rid of the variant-containing plasmid, as seen with the clear
difference in lag phase between the selective and permissive
growth condition (U18C+ and U18C"; Figure 5F). These observations hence demonstrate that kinetically delaying folding
transitions for SRP RNA to attain its functional native fold inturn modulates cell growth.
DISCUSSION
Using a broad set of tools, from optical tweezers to in vitro andin vivo assays, we uncover how the co-transcriptional folding
pathway of SRP RNA directs the attainment of its functional
native form. An obligatory folding intermediate adopted during
early transcription both enables the maturation of SRP RNA
and resists alternative structural rearrangements to secure theattainment of the native form and cellular functionality. Specifically, the non-native intermediate H1 of SRP RNA persists
throughout most of the co-transcriptional folding trajectory until
the RNA 30 end emerges and competes to anneal with the RNA
50 end sequestered in H1, forming the activity-essential distal
stem in the final long-hairpin fold of SRP RNA. Toehold-mediated
RNA strand displacement is a thermodynamically feasible mechanism for the RNA 30 end single strand to invade and resolve H1(Zhang and Winfree, 2009). During toehold invasion, various
stretches of the 30 single-strand sample alternative base-pairing
interactions with the loop region of H1 until a strong enoughhybridization is established, compensating for the entropy
cost of bringing the RNA ends together, to eventually attain complete strand displacement. The large-scale hopping dynamics
observed in the co-transcriptional folding trajectory (Figure 2B)
most likely reflect those transient toeholding attempts between
the two ends on SRP RNA. Finally, the folding trajectories presented here have been obtained in vitro and, therefore, it ispossible that some of the details of these trajectories may be
modified in vivo through interactions with other cellular components. Nonetheless, we note that the predictions derived fromthe single-molecule in vitro experiments about the possible effects of SRP RNA mutants on cell viability are indeed validated
in the cell experiments, suggesting that the in vivo milieu does
not alter significantly the essential features of the folding trajectories presented here.
Future work may elucidate the present or vestigial adaptive
advantage of the SRP RNA 50 precursor, which does not alter
the folding trajectory and can be removed during early transcription. Furthermore, the robust folding pathway for SRP
RNA, pacing its maturation and directing the proper attainmentof its native fold, suggests an elegant strategy to devise RNAs
whose functionalities (such as riboswitches, transcriptionalterminators, and pausing elements) evolve over time by cotranscriptionally transitioning through sequential functional
folds. We also envision that co-transcriptional modulation onPlease cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

RNA folding and function in other essential RNAs could be targeted by antisense therapeutics to further regulate cellular
processes.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITYDECLARATION OF INTERESTS
The authors declare no competing interests.
Received: August 18, 2018
Revised: August 29, 2019
Accepted: October 4, 2019
Published: November 6, 2019SUPPORTING CITATIONS
The following reference appears in the Supplemental Information: Chenet al. (2013).

d METHOD DETAILSB Optical Tweezers
B SRP RNA Encoded Template DNA
B DNA Handle
B Bead Surface Functionalization
B Recombinant Proteins
B Real-Time Co-transcriptionalFolding of Nascent RNA
B Force-Extension (F-X) Curve Measurements of Trun-cated and Full-Length SRP RNA on the Surface of
RNA Polymerase
B Force-Extension (F-X) Curve Measurements of Truncated and Full-Length SRP RNA in Isolation
B Unit Conversion of Transcription Trajectories Recorded in Nanometers into Nucleotides
B RNase P Processing Assay
B Plasmid Constructs for Cell Viability Experiments
B CompletePlasmid
SequenceofpZE21_wtSRP_FL_rrnC
B Cell Viability Plate Assay
B Recovery/Post-Relaxation Growth Rated QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY

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SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
molcel.2019.10.006.

ACKNOWLEDGMENTS
We thank L. Alexander for critical reading of the manuscript, L. Wee for sharing
expertise on biochemistry assay developments, and all members of Bustamante laboratory for discussion. We also acknowledge J. Lucks, A. Yu, andE. Strobel at Northwestern University and V. Gopalan and L. Lai at Ohio State
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AUTHOR CONTRIBUTIONS
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STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Sigma-Aldrich

Cat# 11333089001; RRID: AB_514496

DH5a competent E. coli cells

Thermo Fisher

Cat# 18-265-017

DH5a competent E. coli cells, PN25-tetR

EXPRESSYS

Cat# DH5alpha-Z1

BL21 competent E. coli cells, non-T7 expression

NEB

Cat# C2530H

BL21 competent E. coli cells, Rosetta2 (DE3) pLysS

Macrolab, QB3-Berkeley

N/A

Antibodies
Anti-digoxigenin (from sheep)
Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins
NTP set, 100 mM solution

Thermo Fisher

Cat# R0481

ApU RNA Dinucleotide (50 -30 )TriLink

Cat# O-31004

GpC RNA Dinucleotide (50 -30 )TriLink

Cat# O-31010

Sulfo-NHS (N-hydroxysulfosuccinimide)

Thermo Fisher

Cat# 24510

EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)

Thermo Fisher

Cat# 77149

Heparin sodium salt

Sigma-Aldrich

Cat# H3149

Kanamycin sulfate

GoldBio

Cat# K-120-25

SYBR Green II RNA gel stain

Invitrogen

Cat# S7564

RNaseOUT

Invitrogen

Cat# 10777019

SUPERase In RNase inhibitor

Invitrogen

Cat# AM2694

Biotinylated short peptide, GGGGDGDY{K(biotin)}

Genscript

Custom orderb-casein

Sigma-Aldrich

Cat# C6905

Bovine Serum Albumin (BSA)

NEB

Cat# B900S

Streptavidin

NEB

Cat# N7021S

Turbo DNase

Invitrogen

Cat# AM2238

SuperScript IV reverse transcriptase

Invitrogen

Cat# 18090050

T4 DNA ligase

NEB

Cat# M0202L

Quick Ligation kit

NEB

Cat# M2200

Restriction enzyme, BbsI

NEB

Cat# R3539

Restriction enzyme, AvrII

NEB

Cat# R0174

Phusion! Hot Start Flex DNA polymerase

NEB

Cat# M0535S

Vent (exo-) DNA polymerase

NEB

Cat# M0257S

E. coli RNA polymerase, Holoenzyme

NEB

Cat# M0551S

Biotinylated RNA polymerase

Gabizon et al., 2018

N/A

GreB, E. coli

Gabizon et al., 2018

N/A

Sigma 70, E. coli

Gabizon et al., 2018

N/A

TEV protease

Tropea et al., 2009

N/A

SortaseChen et al., 2011

N/A

RNase P (M1 RNA + C5 protein), E. coli

Gopalan et al., 1997

N/A

Q5 site-directed mutagenesis kit

NEB

Cat# E0554

QIAquick PCR purification kit

QIAGEN

Cat# 28104

Critical Commercial Assays

Oligonucleotides
Lambda DNA

NEB

Cat# N3011S

bead cross-link oligo: 50 -/5AmMC6/TTAATTCATTGCGTTCTGT
ACACG-30

IDT

custom order(Continued on next page)

Molecular Cell 77, 1–10.e1–e8, January 16, 2020 e1

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

bead cross-link complementary oligo: 50 -/5Phos/CGGTCGTGTACAGAACGCAATGAATT-30

IDT

custom order

gblock for mature_SRP RNA: 50 -AGTCTAACCTATAGGATACTTA
CAGCCATCCCACCACAACCACCACAACCACCACAACAACAACCAATCGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCAC
CCCAACAAGAAACCACACAACAACACCAACAACAAACAGACGAATTCTCATCACCATCATCCTGACTAGAG-30

IDT

custom order

gblock for pre_SRP RNA: 50 -AGTCTAACCTATAGGATACTTACAGCCATCCCACCACAACCACCACAACCACCACAACAACAAC
ACCGCGTTGGTTCTCAACGCTCTCAATGGGGGCTCTGTTG
GTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCCCAACAAGAAACCACACAACAACACCAACAACAAACAGACGAATTCTCATCACCATCATC
CTGACTAGAG-30

IDT

custom orderforward primer for template DNA in real-time transcription
experiments: 50 -CAGATCCCGAACGCCTATC-30

IDT

custom orderreverse primer for template DNA in real-time transcription
experiments: 50 -GTCTGTTTGTTGTTGGTGTTGTTG-30

IDT

custom orderbiotinylated primer_40 bp for F-X curve measurements:
50 -/5Biosg/GTAAACAGAGTAGCGTTGCG-30

IDT

custom orderbiotinylated primer_52 bp for F-X curve measurements:
50 -/5Biosg/GGACCTGACCTGGTAAACAG-30

IDT

custom orderbiotinylated primer_84 bp for F-X curve measurements:
50 -/5Biosg/ACACGCGTCATCTGCC-30

IDT

custom orderbiotinylated primer_97 bp for F-X curve measurements:
50 -/5Biosg/GCTACATCCCGGCACAC-30

IDT

custom orderbiotinylated primer_114+16 bp for F-X curve measurements:
50 -/5Biosg/GTGTGGTTTCTTGTTGGGGTG-30

IDT

custom orderbiotinylated primer_114+39 bp for F-X curve measurements:
50 -/5Biosg/GTCTGTTTGTTGTTGGTGTTGTTG-30

IDT

custom orderantisense oligo-1: 50 -CAGAGCCCCC-30

IDT

custom orderantisense oligo-2: 50 -GACCTGACCTGG-30

IDT

custom order

gblock for T7A1_4.5S RNA: 50 -GGAAACAGCTATGACCATGATTACGCCAAGCTTAAAAGATTAATTTAAAATTTATCAAAAAGAGTATTGACTTAAAGTCTAACCTATAGGATACTTACAGCCGCGTTG
GTTCTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGC
AACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCA
GCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCA
GGGCCCCCACCCATTTCTGCCTCCCACCGTTTCGTCAAAAAATCCCAACATGGCTAAACT-30

IDT

custom ordercommon forward primer for stalling template DNA:
50 -GGAAACAGCTATGACCATGATTACGCC-30

IDT

custom orderbiotinylated reverse primer-1 for stalling template DNA(24+22): 50 -CCGGACC/iBiodT/GACCTGGTAAACAG-30

IDT

custom orderbiotinylated reverse primer-2 for stalling template DNA(24+30): 50 -GCTTCCT/iBiodT/CCGGACCTGACC-30

IDT

custom orderbiotinylated reverse primer-3 for stalling template DNA(24+66): 50 -CTACA/iBiodT/CCCGGCACACGC-30

IDT

custom orderbiotinylated reverse primer-4 for stalling template DNA(24+114): 50 -CCATGTTGGGATTTTT/iBiodT/GACGAAACGGTG-30

IDT

custom order(Continued on next page)e2

Molecular Cell 77, 1–10.e1–e8, January 16, 2020

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

cDNA primer for reverse transcription: 50 -/5TYE665/
GTAAACAGAGTAGCGTTGCGGGAG-30

IDT

custom order

gblock for pZE21_4.5S_rrnC: 50 -CACGAGGCCCTTTCGTCTTCAC
TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAG
ATACTGAGCACGCGTTGGTTCTCAACGCTCTCAATGGGGGCT
CTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGG
TCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGG
GATGTAGCTGGCAGGGCCCCCACCCCAGATAAAAAAAATCC
TTAGCTTTCGCTAAGGATGATTTCTACCTAGGGCGTTC-30

IDT

custom order

Recombinant DNA
pZ vector (pZE21MCS)

EXPRESSYS

Cat# pZE21MCS

pZE21_wtSRP_FL_rrnC

This paper

N/A

pZE21_SRP_100_rrnC

This paper

N/A

pZE21_U18C_FL_rrnC

This paper

N/A

pZE21_U18C_100_rrnC

This paper

N/A

pZE21_U3234C_FL_rrnC

This paper

N/A

pZE21_U3234C_100_rrnC

This paper

N/A

PGMZ plasmidThis paper

N/A

pIA1127

This paper

N/A

pIA1234

This paper

N/A

Software and Algorithms
LabVIEW

National Instruments

N/A

MATLAB

Mathworks

N/A

Igor Pro

WaveMetrics

N/A

Other
Mono Q 5/50 GL, anion exchange chromatography column

GE Healthcare

Cat# 17-5166-01

HiPrep 16/60 Sephacryl S-300 HR Column

GE Healthcare

Cat# 17-1167-01

Carboxylated polystyrene beads, 1-mm

Bang Laboratories

Cat# PC04N

Streptavidin coated polystyrene beads, 0.82-mm

Spherotech

Cat# SVP-08-10

LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Carlos Bustamante(carlosb@berkeley.edu).
METHOD DETAILS
Optical Tweezers
A home-built optical tweezers instrument was assembled as described previously (Gabizon et al., 2018; Righini et al., 2018). Briefly,
an Nd:YAG 1,064-nm laser was used. The two traps were generated by switching a single beam at high rate using acoustic-optic
deflector (AOD). The frequency of the AOD was controlled by a custom-made radio frequency board (Comstock et al., 2011), andthe position of the traps were switched every 5 ms. In this case, detection of the bead positions in both traps was achieved on thesame quadrant photodiode (QPD).
SRP RNA Encoded Template DNA
A DNA fragment (IDT) coding for the Escherichia coli (E. coli) SRP RNA (138-bp for pre-SRP RNA, or 114-bp for mature SRP RNA) was
first cloned into a PGMZ plasmid at 42-bp downstream of T7A1 promoter. Specific length of template DNAs—either full-length forreal-time transcription experiments or truncated versions for nascent RNA folding intermediate unfolding assessments—was thenamplified by PCR (using different reverse primers, IDT) from the cloned plasmid. The short G-less sequence placed downstreamof the T7A1 promoter allows us to stall the RNAP transcription right before the beginning of SRP RNA, which we will then restarton the optical tweezers setup by supplementing the full set of NTPs (see main text). Also, it is with this stretch of initially transcribedstalled sequence on the nascent RNA 50 end that a 50 overhang bearing dsDNA handle anneals so as to catch the RNA tether on the
Molecular Cell 77, 1–10.e1–e8, January 16, 2020 e3

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

optical tweezers. To prepare DNA templates for truncated SRP RNA folding intermediates stalled on the RNAP surface (Figure S3),
the corresponding PCR reactions were performed with biotinylated reverse primers (IDT).
DNA Handle
The DNA handles were prepared by PCR using lambda DNA as the template, and with biotinylated or digoxigenin-labeledoligonucleotides (IDT) as primers (right- and left-hand side of the DNA handles shown in Figure 2A), purified following standard protocol from PCR purification kit (QIAGEN). To create the DNA handle bearing a 50 overhang complementary to the nascent RNA 50 end,
a 1’, 20 -Dideoxyribose was introduced in the DNA primer (IDT) and used in the PCR reaction.
Bead Surface Functionalization
Anti-digoxigenin (AD) antibody coated beads
50 mL of 5% (w/v) 1-mm diameter carboxylated polystyrene beads (Spherotech) were washed with 1 mL activation buffer (100 mM
MES-NaOH, pH 6, 500 mM NaCl) three times, with centrifugation steps (3 min at 3000 rpm) between wash steps. EDC and sulfoNHS (Thermo Scientific) were then added to the bead solution to final concentration of !5 mM and !10 mM, respectively. Afterincubation for 15 min at room temperature, the reaction was quenched by adding 2.8 mL 2-mercaptoethanol (40 mM). After washing
the beads with activation buffer, the anti-digoxigenin antibody was incubated with bead solution by tumbling at room temperature.
The reaction proceeded for 2 hours. The beads were washed with quenching buffer (100 mM Tris-HCl pH 7) and storage buffer(40 mM HEPES-KOH, pH 7.5, 100 mM KCl), and stored at 4# C.
Oligo coated beads
To prepare a double stranded oligo for bead surface coupling reaction, 50 end amino modified DNA oligo (50 -/5AmMC6/TTAATTCATTGCGTTCTGTACACG-30 , IDT) was hybridized to a 50 -phosporylated DNA oligo (50 -/5Phos/CGGTCGTGTACAGAACGCAATGAATT-30 , IDT). Annealing was performed by heating the mixture of DNA oligo (0.25 mM each) to 95# C for 10 min, followed by coolingto room temperature. 10 mL of 10% (w/v) 1 mm-diameter carboxylated polystyrene beads (Spherotech) were washed four times withcoupling buffer (100 mM MES-NaOH pH 4.7, 150 mM NaCl, 5% DMSO), and spun down (5 min at 4500 g) between washes. The
annealed double stranded oligo and EDC (Thermo Scientific Pierce) were then added to the washed beads to a final concentrationof !1.5 mM and !300 mM, respectively. After incubation for 2 hours with vigorous shaking, more EDC (final concentration !500 mM)
was added, followed by overnight shaking at room temperature. The reaction was quenched upon the addition of glycine (final concentration !50 mM). Lastly the bead solution was washed with stock buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA, 0.05% Tween 20,
5 mM NaN3) four times and stored at 4# C.
Beads passivation
The above functionalized beads (1%, w/v) were passivated by diluting 10-fold in TE buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA) andaddition of casein to a final concentration of 1 mg/ml. Then the bead sample was incubated with vortex for !1 hour at room temperature and washed with TE buffer by centrifugation (3 min at 4500 g), and stored at 4# C. 0.82-mm diameter streptavidin coated beads(Spherotech) were also passivated following the same protocol.
Recombinant Proteins
Preparation of sigma 70
Plasmid pIA1127 was transformed into Rosetta2 bacteria. The bacteria were grown in 2 L of 2YT medium supplemented with 1%
glucose, NPS (25 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4), 1 mM magnesium sulfate, 34 mg/ml chloramphenicol, and
50 mg/ml kanamycin. The culture was grown at 37# C and transferred to 17# C. Then IPTG was added to 0.1 mM and incubated for
16 hours. For purification, the bacteria were dispersed in 80 mL of buffer A25 (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 10% glycerol,
25 mM imidazole, 2 mM beta-mercaptoethanol) supplemented with 0.1 mg/ml lyzozyme and protease inhibitors (Roche). The bacteria were then lysed via French press, and the lysate was clarified by centrifugation and filtration and loaded onto a 5 mL Ni-NTA
column. The column was washed with 20 mL buffer A25 and 20 mL buffer A50 (A25 + 50 mM imidazole), and the his-tagged sigma
70 was eluted in buffer A300 (A25 + 300 mM imidazole). For the removal of the his-tag on sigma 70, TEV protease (Tropea et al., 2009)
was added to the eluted protein at a molar ratio of 1:40, and reacted overnight at 4# C while being dialyzed against A50. The protein
was then again passed through a Ni-NTA column. The flow-through, containing non–his-tagged sigma 70 was collected,
concentrated and purified by gel filtration on a sephacryl S300 column equilibrated with buffer B (20 mM Tris, pH 7.5, 0.5 M NaCl,
10% glycerol, 1 mM EDTA, 1 mM DTT). Aliquots were flash-frozen in liquid nitrogen and stored at "80# C.
To form the holoenzyme, the sample was incubated with a twofold excess of purified sigma 70 overnight on ice and diluted 10-foldwith buffer B0 (50 mM Tris-HCl, pH 6.9, 5% glycerol, 0.5 mM EDTA, 1 mM DTT) and loaded on a heparin 5 mL column. A gradient of
50 mM to 1 M NaCl was used to elute the protein. RNAP holoenzyme was separated clearly from excess sigma 70. The sample wasdialyzed against buffer B50 (50 mM Tris-HCl, pH 6.9, 5% glycerol, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT) and then purified further on
a 1 mL monoQ column using a 50 mM to 1 M NaCl gradient (again, the sample was split into three portions loaded separately). Purefractions were pooled, dialyzed against storage buffer (20 mM Tris-HCl, pH 7.5, 200 mM KCl, 0.2 mM EDTA, 0.2 mM DTT, 5%
glycerol), aliquoted, flash-frozen, and stored at "80# C.

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Molecular Cell 77, 1–10.e1–e8, January 16, 2020

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Preparation of sortagged RNA polymerase holoenzyme
Plasmid pIA1234 was transformed into Rosetta2 bacteria. Sortag-RNAP was expressed using the same protocol as sigma 70, exceptthat ampicillin was used instead of kanamycin. For purification, the cells were dispersed in 75 mL of lysis buffer (50 mM Tris-HCl, pH
6.9, 0.5 M NaCl, 5% glycerol) supplemented by 0.1 mg/ml lyzozyme and protease inhibitors and lysed via French press. The lysate
was centrifuged and filtered, and imidazole was added to 20 mM. The protein was loaded onto a 5 mL Ni-NTA column. The column
was washed with 30 mL of lysis buffer plus 20 mM imidazole, and the his-tagged RNAP core enzyme was eluted in lysis buffer plus
250 mM imidazole. To form the holoenzyme, the sample was incubated with a twofold excess of purified sigma 70 overnight on ice
and diluted 10-fold with buffer B0 (50 mM Tris-HCl, pH 6.9, 5% glycerol, 0.5 mM EDTA, 1 mM DTT) and loaded onto a heparin 5 mLcolumn. A gradient of 50 mM to 1 M NaCl was used to elute the protein. RNAP holoenzyme was separated clearly from excess sigma
70. The sample was dialyzed against buffer B50 (50 mM Tris-HCl, pH 6.9, 5% glycerol, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT) andthen purified further on a 1 mL monoQ column using a 50 mM to 1 M NaCl gradient (again, the sample was split into three portions
loaded separately). Pure fractions were pooled, dialyzed against storage buffer (20 mM Tris-HCl, pH 7.5, 200 mM KCl, 0.2 mM EDTA,
0.2 mM DTT, 5% glycerol), aliquoted, flash-frozen, and stored at "80# C.
Biotinylation of sortag-RNA polymerase
We obtained a peptide containing an N-terminal GGG tag with a biotin-modified lysine residue (Genscript): GGGGDGDY{K(biotin)}.
We reacted 100 mL of 9.6 mM sortag-RNAP with a 200-fold excess of biotinylated peptide in 200 mL coupling buffer (50 mM Tris-HCl,
pH 7.5, 5 mM CaCl2) containing 2 mM sortase (Chen et al., 2011). The reaction proceeded for 60 min. At this point, imidazole wasadded to 25 mM and NaCl to 350 mM, and the sample was passed through 70 mL Ni-NTA beads to remove sortase and unreacted
RNAP. The excess peptide was removed by dialysis into storage buffer, and the biotinylated RNAP was stored in storage buffer
at "80# C.
Preparation of GreBGreB was purified as described previously (Gabizon et al., 2018).
Real-Time Co-transcriptionalFolding of Nascent RNA
Stalled RNAP-RNA complexes were prepared by incubating biotinylated RNAPs with template DNAs in TB20 buffer (20 mM Tris-HCl,
pH 8, 20 mM NaCl, 10 mM MgCl2, 20 mM DTT, 1 mg/ml BSA) containing 5 mM ATP, CTP, 150 mM ApU and RNaseOut (ThermoFisher)for 20 minutes at 37# C. Stalled complexes were then incubated for another 20 min together with heparin and digoxigenin-labeled
1.5-kbp DNA handle bearing a 50 overhang complementary to the 50 stretch of nascent RNA transcripts. 1-mm diameter anti-digoxigenin coated (AD) beads were then added to the stalled complexes. After 20 min incubation, the mixture was diluted into 1 mL TB130
buffer (20 mM Tris-HCl, pH 8, 130 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT and 10 mM NaN3). Meanwhile, 1-mm diameter
oligo-coated beads were ligated to the 50 overhang of another 1.5-kbp DNA handle which is biotin-labeled on its other end (Figure 2A);
the ligation reaction is carried out in TB20 buffer including 0.4 units of T4 DNA ligase and 0.1 mM ATP for 1 hour at room temperature.
Following the ligation, the handle-bead mixture was incubated with streptavidin for another 20 min and finally diluted into 1 mL TB130
buffer. Next the stalled complex bead and handle-bead samples were delivered into the microfluidic chamber on the optical tweezers
setup and trapped by the two laser beams, respectively. The two beads were then brought to close proximity of each other, such thata tether could form between the streptavidin bound on the 1.5-kbp DNA handle and the biotin labeled on the RNAP (Figure 2A). Oncea stalled RNAP:RNA complex is tethered on the optical tweezers setup, the synthesis of nascent transcript is fueled by flowing TB130
buffer containing NTPs into the chamber, where the extension/growth of the RNA is monitored under constant force. For real-timetranscription experiments conducted in the presence of GreB or antisense oligo, the transcription buffer was further supplemented
with 1 mM of GreB, or 10 mM HPLC-purified antisense oligo (IDT).
Force-Extension (F-X) Curve Measurements of Truncated and Full-Length SRP RNA on the Surface of RNAPolymerase
As a roadblock to stall RNAP on the template DNAs of different length—so as to transcribe specific length of nascent SRP RNA transcript (Figure S3), a streptavidin was bound to the biotin labeled at the downstream end of the template DNA; the mixture is incubatedin TB20 buffer at room temperature for 1 hour. Stalled complexes were then prepared by incubating DNA:streptavidin complex withRNAP in TB20 buffer containing 5 mM ATP, CTP, 150 mM ApU and RNaseOut (ThermoFisher) for 20 minutes at 37# C. Stalled
complexes were then incubated for another 20 min together with heparin and digoxigenin-labeled 1.5-kbp DNA handle bearing a
50 overhang complementary to the 50 stretch of nascent RNA transcripts. 1-mm diameter anti-digoxigenin coated (AD) beads werethen added to the stalled complexes; all procedures up till now are the same as those in the above real-time stalled complex
preparation. After 20 min incubation, the mixture was incubated with NTPs (!50 mM) for 1 min to further transcribe SRP RNA
from the template DNA, and finally diluted into 1 mL TB130 buffer. Meanwhile, the handle-bead sample was preparedfollowing the same ligation protocol as that used in the real-time transcription experiments, but diluted into TB130 buffer
without further incubation with streptavidin. In this case, a stalled RNAP:RNA complex is tethered on the optical tweezers setup
via the binding interaction between the streptavidin at the downstream end of template DNA and the biotin labeled on the
1.5-kbp DNA handle.

Molecular Cell 77, 1–10.e1–e8, January 16, 2020 e5

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

Force-Extension (F-X) Curve Measurements of Truncated and Full-Length SRP RNA in Isolation
A purified SRP RNA was incubated with digoxigenin-labeled 1.5-kbp DNA handle bearing a 50 overhang complementary to the 50
stretch of RNA in TB20 buffer for 20 minutes. 1-mm diameter anti-digoxigenin antibody coated (AD) beads were then added toRNA-handle complexes. After 20 min incubation, the mixture was diluted into TB130 buffer. For the other side of the tethering beads,
0.82-mm diameter streptavidin coated beads were incubated with a biotin-labeled 1.5-kbp DNA handle bearing a 30 overhang (Yanet al., 2015) complementary to the 30 stretch of RNA in TB20 buffer for 20 min, and the mixture was diluted in TB130 buffer. Tethering
between the 1-mm and 0.82-mm beads trapped on the optical tweezers setup was made via the complementary sequences of RNA 30
stretch and the biotin-labeled DNA handle 30 overhang.
Unit Conversion of Transcription Trajectories Recorded in Nanometers into Nucleotides
In order to convert the observed transcription extension recorded in nanometers into nucleotides, we consider the force-dependent
characteristics determined from the extensible worm-like chain (WLC) model (Bustamante et al., 1994):
FP 1!
x ""2 1 x F(Equation 1)
= 1"
" + "
kB T 4L
4 L K

where kB is the Boltzmann constant, K is the elastic modulus, L is the counter length, and P is the persistence length, which represents the stiffness of RNA and greatly impacts the conversion factor between nanometers and nucleotides. It has been shown thatpersistence length is altered by buffer conditions such as ionic strength (Chen et al., 2012), where changes in [NTP] under fixed [Mg2+]
affect the RNA extension change observed at the same constant force on the optical tweezers. To determine the persistence length inour experimental conditions, we measure the unfolding/refolding extension change in nanometer of a known RNA hairpin which is180-bp long (Yan et al., 2015), and calculate the conversion factors (nt/nm) under specific constant force applied and at a given[NTP] (see Table S1). These empirically determined values were also verified using the full-length SRP RNA (in the absence of
RNAP), which readily adopts its 50-bp long native hairpin fold.

RNase P Processing Assay
Four stalled RNAP-RNA complexes (see main text) were prepared using the aforementioned streptavidin roadblock method to stall
RNAP on a biotinylated template DNA, where the biotin is located !10-nt downstream of the desired stalled site and is on the template strand to achieve high stalling efficiency (Strobel et al., 2017). Specifically, 4.5 pmole of biotinylated DNA template (PCR
products using Vent (exo-) DNA polymerase from NEB, and primers with internal biotin modifications from IDT; gel-purified) was
pre-incubated with 225 pmole streptavidin (NEB) at room temperature for 30 min (total volume 18.75 ml; in 1x E. coli RNA polymerase
reaction buffer; M0551, NEB). Meanwhile, 1.5 mL of 3 mM C5 protein is pre-incubated at 37# C for 5 min, and then incubated with 1.5 mL
of 0.6 mM M1 RNA for another 10 min at 37# C to form the RNase P complex (gift from Dr. Lien Lai and Prof. Venkat Gopalan) (Gopalan
et al., 1997; Gössringer et al., 2012; Vioque et al., 1988). Next, 16.5 mL reaction mixtures containing 9.375 mL DNA-roadblock template(final 136 nM), 1.5 mL RNase P complex (final 27 nM; or 1.5 mL 1x buffer for control), and 5.625 mL of RNAP solution (final: 0.20U/ml
RNAP; M0551, NEB; 0.45 mM NTP; 0.04 mM GpC, TriLink; 0.3U/ml SUPERase,In, Invitrogen; 1x reaction buffer) were incubated at
37# C for 10 min. 1.1 mL of Turbo DNase (Ambion) is then added to the above reaction to digest away the DNA template (37# C for
15 min). 8 mL of the above final mixtures were quenched with equal volume of gel loading buffer (final 49.5% formamide, 9 mM
EDTA; Ambion) and loaded onto Urea PAGE gel (7M, 12%, Figure 4B) to visualize the RNA products. The other 8 mL is mixed with
1 mL glycogen (0.5 mg/ml) prior to phenol/chloroform extraction and ethanol precipitation, so as to concentrate the RNA products
for reverse transcription of cDNA with a 50 -TYE665-labeled DNA oligo primer (IDT) using SuperScript IV (Invitrogen). Half of the above
reaction is loaded onto Urea PAGE gel to visualize the cDNA products (in the red fluorescence channel; Figure 4C) as well as theRNAs (SYBR Green II staining). The other half of the cDNA products, after phenol/chloroform extraction and ethanol precipitation,is ligated (T4 DNA ligase; M0202, NEB) to a donor DNA oligo for PCR amplification (Phusion DNA polymerase; M0535, NEB) (Nat.
Methods, 2005; Guo et al., 2010; Moore and Query, 2000). The resultant DNA products were then gel-purified, where individual bands
were eluted separately and sent for sequencing (Sequetech).
Plasmid Constructs for Cell Viability Experiments
pZE21MCS (MCS: multiple cloning sites; sold by Expressys; a gift from A. Flamholz) and customized gblocks (!500 bp long
double-stranded DNAs, which sequence from 50 to 30 contains: PLtetO-1 promoter, specific SRP RNA variant sequence, rrnC
terminator, and a second PLtetO-1; purchased from IDT) were digested with BbsI and AvrII (R3539 and R0174, NEB), ligated with quick
T4 ligase kit (M2200, NEB) following NEB protocol, and transformed into an E. coli cloning strain, DH5alpha-Z1 (Expressys). A seriesof plasmids (pZE21_SRP_rrnC) were constructed: pZE21_wtSRP_FL_rrnC, pZE21_SRP_100_rrnC, pZE21_U18C_100_rrnC,
pZE21_U3234C_100_rrnC, pZE21_empty_rrnC, and pZE21_SRP_100_rrnC_read-through. The plasmids harboring full-length SRP
RNA sequence mutants (U18C and U32C/U34C) were prepared from pZE21_wtSRP_FL_rrnC using the Q5 site-directed mutagenesis kit (E0554, NEB), and following protocols therein for appropriate primer designs and sequential procedures to obtain the modified plasmids (pZE21_U18C_FL_rrnC and pZE21_U3234C_FL_rrnC).
Since the 24-nt long 50 -precursor sequence in the pre-SRP RNA (i.e., 4.5S RNA) does not interfere with the co-transcriptional
folding of SRP RNA (see main text), it is not included in these plasmid constructs prepared for our cell viability experiment. Leaving

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Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

out the 50 -precursor also eliminates the possibility that the maturation, instead of the proper folding, of SRP RNA is the cause for any
changes seen with cell viability. This potential complication is due to the fact that RNase P—the processing enzyme that cleaves off
the 50 -precursor from SRP RNA—is five times less abundant than the endogenous pre-SRP RNA (Dong et al., 1996), and would beeven more limiting if it were to process an exceeding amount of exogenous pre-SRP RNA variants that are highly transcribed inside
the transformed cells. Furthermore, RNase P also processes many other essential functional RNAs such as tRNAs. In other words,
any shortage (or sequestering) of this processing enzyme will inevitably impact the overall cell viability and compromise the interpretation of our analysis. Therefore, to better and directly correlate how the folding of SRP RNA influences its functionality and ultimately
the cell viability, we choose to not include its 50 -precursor in the plasmid constructs prepared for our in vivo studies.
The fact that the same promoter sequence (i.e., PLtetO-1) is used to regulate the transcription of desired RNA products, and that thecloning plasmid is of high copy number (i.e., colE1 replication origin; 70-100 plasmid copy number/cell), ensures similar overexpression levels of the SRP RNA variants examined in the cell experiments. We have verified that similar amounts of plasmid are indeed
maintained in the bacteria. Specifically, at each round of cell growth experiment, we isolated the plasmids from transformed cells (ofthe same cell density [OD600] and culture volume) to confirm the yield of the plasmids as well as the propagation of the correct
sequence for each SRP RNA variant. To verify the same promoter indeed behaves the same for different variants inserted downstream in the plasmid, we have also performed in vitro transcription with the isolated plasmids and found similar yields in the RNA
products transcribed (data not shown). Based on the above information and characterizations, we can attribute the observed impacts
on the cell viability—not to the differences in expression levels but—to the changes in RNA folding of different SRP RNA variants
examined.
CompletePlasmid Sequence of pZE21_wtSRP_FL_rrnC
Please see the last two pages in the Supplemental Data file for detailed notations on the sequence elements and specific features
highlighted within the plasmid.
50 -CTAGGCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC
CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT
GGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA
GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA
ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGACTAGTGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGC
GATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGG
CATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCC
CTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTG
GTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAG
CAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTG
TGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGG
GCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCCCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTCC
TCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCGGATCCCATGGAGGCATTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAATCTAGACAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTAC-30
Cell Viability Plate Assay
BL21 (non-T7 expression, C2530H from NEB) were transformed with purified, sequencing-verified plasmids (pZE21_SRP_rrnC) thatcarry specific SRP RNA variants (following protocol from NEB; 50 ng plasmid per 50 mL competent cell) and plated on kanamycin(40 mg/ml) LB agar plates. Images of the plates, showing the emergence of colonies that carry plasmids encoding with specific

Molecular Cell 77, 1–10.e1–e8, January 16, 2020 e7

Please cite this article in press as: Fukuda et al., The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.10.006

SRP RNA variants, were taken after 12-16 hours of growth at 37# C. At least 5 colonies were picked from each transformation experiments and sent for sequencing (Sequetech) to confirm the plasmid constructs therein.
Recovery/Post-Relaxation Growth Rate
BL21 (non-T7 expression, C2530H from NEB) were transformed with purified, sequencing-verified plasmids (pZE21_SRP_rrnC) thatcarry specific SRP RNA variants (following protocol from NEB; 50 ng plasmid per 50 mL competent cell) and plated on kanamycin(40 mg/ml) LB agar plates. A single colony was picked and inoculated into 10 ml of LB (antibiotic-free), and let grow at 37# C withshaking at 180 rpm for 6 hours. We then split each culture sample in half, where 5 mL continues to grow in LB (i.e., permissive condition), and the other 5 mL grows in the presence of kanamycin (final conc. 40 mg/ml), for another 10 hours. After a total of 16 hours(6+10) overnight growth, aliquot from each culture is diluted with LB plus kanamycin (final conc. 40 mg/ml) to OD600 = 0.02 as thestarting point of recovery growth (t0). We then record OD600 every 30-45 min interval, to monitor the rate of growth at 37# C, for a
total of 3-4 hours until the culture reaches the mid-log phase (OD600 = 0.6-0.8).
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical details of individual experiments, including numbers of observations, numbers of replicates, and dispersion and precision
measures, are as described in the manuscript text, figure legends, and the figures themselves.
DATA AND CODE AVAILABILITY
The full dataset will be made available upon request.

e8

Molecular Cell 77, 1–10.e1–e8, January 16, 2020

A

E

B

F

C

D

E

Figure S1F

Figure S1. Characterizing the Mean Durations of Transcription Pauses and MeanTime Span After Pause in the SRP RNA Co-transcriptional Folding Trajectories: asa Function of [NTP], of the Presence and Absence of the Precursor Sequence, and ofSpecific Nucleotide Depletion; Related to Figure 2(A) Mean durations for P1 pause (left), mean durations for P2 pause (middle), and mean time span afterP2 pause (right; corresponding to purple regions in Figure 2B) in the SRP RNA co-transcriptional
folding trajectories, which were recorded in the presence and absence of the 5’-precursor sequence(filled and empty bars, respectively), and at three NTP concentrations: 1 mM (n=14, 11), 50 μM (n=14,14), and 10 μM (n=13, 12).
(B) Representative traces of SRP RNA co-transcriptional folding at [UTP] = 10 μM (1 mM each for
ATP, CTP, and GTP). Both P1 and P2 pauses are prolonged.
(C) Representative traces of SRP RNA co-transcriptional folding at [GTP] = 10 μM (1 mM each for
ATP, CTP, and UTP). Only P2 pause duration is prolonged.
(D) Representative traces of SRP RNA co-transcriptional folding at low [CTP] (orange) or low [ATP](magenta) ([CTP] or [ATP] = 10 μM, while the others are at 1 mM). The trace at saturating [NTP] (1mM each; black) is shown for comparison.
(E-F) Mean durations for P1 and P2 pauses at saturating [NTP] (n=11), low [UTP] (n=10), low [GTP](n=12) transcription conditions, and in the presence of 1 μM GreB (n=10).

A

E

B

1

2

3

4

1

7

F
C

G

WT_114

U32C/U34C_114

FUnf

FUnf

U18C_114

FUnf

D
2

1

2
1

1

8

4

FUnf (pN)

4

Figure S2

FUnf (pN)

H
U18C_100

1

FUnf (pN)

U18C_100

U18C_100

Figure S2. Force-Extension (F-X) Curve Measurements with Nascent RNA:RNAP
Stalled Complexes to Characterize the Folding Progression of SRP RNA; Related to
Figure 2, 3, and 5(A) A schematic cartoon illustrates the folding intermediates of nascent SRP RNA as it is transcribed by
RNAP (same as that shown in Figure 2E). In this study, six stalled RNA:RNAP complexes were
examined. Specifically, the RNAP was stalled by a biotin:streptavidin roadblock located at six different
base-pair positions on the template DNA: 40 bp (cartoon  in Figure 2E), 52 bp (cartoon ), 84 bp(cartoon ), 97 bp (cartoon ), 114+16 bp (cartoon ), and 114+39 bp (cartoon ), so as toincrementally lengthen the nascent RNA chain emerged on the RNAP surface. Since the RNAP footprint
covers ~14-nt on the RNA transcript 3’-end (and ~27 bp on the template DNA), the lengths of RNAsoutside of RNAP are estimated to be ~13-nt (=40-14-27/2; ), ~25-nt (), ~57-nt (), ~70-nt (), and~103-nt (), respectively. For the last complex (), an additional 12 nt (=39-14-27/2) are appended to
the full-length (114-nt) SRP RNA 3’-end, allowing sufficient separation from the RNAP surface.
(B) Representative force-extension (F-X) unfolding curves of incrementally lengthened nascent SRPRNA bound on RNAP: pink for complex , light green for complex , orange for complex , yellow
green for complex , purple for complex  (which can access three competing folding states, as seenfrom the three different unfolding patterns; the relative distribution of those states are evaluated from a
total number of n=153 F-X curves, recorded from 9 molecules), and red for complex , which is a fulllength SRP RNA completely outside of the RNAP. Black arrowheads indicate where one or a series of
unfolding events are detected along the F-X unfolding curves.
(C) Force-extension (F-X) curves (red, unfolding; blue, refolding) of full-length SRP RNA bound on
RNAP (left panel) and of full-length SRP RNA isolated free in solution (right panel). In both cases, thefull-length SRP RNA unfolds in one cooperative transition around 26 pN.
(D) Force-extension (F-X) curve measurements were also attempted co-transcriptionally (i.e. withactively transcribing RNA:RNAP complexes), so as to validate the RNA folding structure assignments
for the nascent SRP RNA under possibly non-equilibrium conditions. Top panel: nascent RNA
unfolding curves measured when transcription progresses from complex  to , i.e. before the initial
hump-like feature and after reaching the P1 pause in the co-transcriptional trace (middle trajectory inblack, F=8.6 pN; see also Figure 2 and main text). The later F-X curve shows a small unfolding rip
signature (black arrowhead), verifying the formation of H1 hairpin upon nascent RNA synthesis.
Bottom panel: nascent RNA unfolding curves measured when transcription progresses from complex
 to , i.e. before the initial hump-like feature and after reaching the P2 pause in the co-transcriptional
trace (middle trajectory in black, F=8.6 pN; see also Figure 2 and main text). The later F-X curve
exhibits two small unfolding rips (black arrowheads), verifying that H1 and H2 hairpins readily form
when half of the SRP RNA nascent chain has emerged from RNAP. The top and bottom sets of co-transcriptional F-X curves were respectively obtained under depleting UTP (10 μM) and depleting GTP(10 μM) transcription conditions, so as to gain time for completing the measurements during the P1 andP2 pauses respectively.
(E) Additional representative F-X unfolding curves measured with complexes , , , , and (same color-code as those shown in panel B) are shown to illustrate the consistency of F-X curvemeasurements across different molecules of the same nascent transcript length, and the reproducibilityof the same molecule with repeating unfolding and refolding cycles. Total number of F-X curves
recorded for complex : n=32 (from 12 molecules); complex : n=40 (from 12 molecules); complex
: n=26 (14 molecules); complex : n=17 (7 molecules); and complex : n=153 (9 molecules).
Complex , which bears a ~100-nt long nearly completed SRP RNA transcript, exhibitsinterchangeable unfolding patterns between unfolding cycles, signifying that this transcript
stochastically populates/samples three accessible folding states. Note that these F-X curves are notdisplayed in the order of consecutive unfolding cycles, but rather in groups of similar unfolding patterns,
so as to provide more examples for the unfolding of complex  unfolding shown in panel B. Thisobservation illustrates that the nearly completed RNA attains competing structural folds, transitioning
between intermediate states and the final long-hairpin native fold (i.e. the right-most unfolding curvethat shows a large single-rip).
(F) Additional representative unfolding F-X curves measured with full-length SRP RNA bound on
RNAP (left plot; total number of curves: n=110, from 10 molecules) and of full-length SRP RNA
isolated free in solution (right plot; total number of curves: n=112, from 13 molecules).
(G) Representative unfolding F-X curves of full-length SRP RNAs (114-nt) containing wild-typesequence (WT_114), or one of the two mutant sequences (U32C/U34C_114 and U18C_114). All threeRNAs unfold cooperatively in one transition, which pattern is consistent with a single long native
hairpin fold as predicted by Mfold. Histograms of unfolding forces (FUnf, marked by gray dash lines)
measured with the respective full-length SRP RNAs listed above are also plotted (from left to right): theaverage unfolding force is 26.2±0.9 pN with WT_114 (total number of curves: n=110, from 12
molecules); 26.2±1.1 pN with U32C/U34C_114 (n=51, recorded from 12 molecules); and 26.1±0.8 pN
with U18C_114 (n=41, recorded from 8 molecules).
(H) Representative unfolding F-X curves of near-completed SRP RNA transcript (100-nt) bearing the
U18C mutation. Three types of unfolding patterns were detected (n=156): 10.2% is of one cooperative
unfolding transition (i.e. a full-length like curve as the left most panel shown in Figure S6A); 19.9% isof two unfolding rips; and 69.9% is of several small consecutive unfolding rips. By contrast, therefolding curves (blue) appear similar and are indistinguishable among the three populations.

A

WT_103

C

B

WT_114D

Figure S3

Figure S3. Step-wise Folding Transition of Nearly-completed vs. Full-length SRP
RNA: Transcript 3’-end Promotes the Attainment of Native Long-hairpin Fold;
Related to Figure 2 and Figure 5
Step-wise folding transitions of wild-type SRP RNA—either nearly completed (103-nt, panel A) or as
full-length (114-nt, panel B)—on the surface of RNAP (see also cartoon 6 & 8 in Figure 2E) arerecorded using the force-clamp optical tweezers assay. In this assay, the tether is held at constantforce and transitions between alternative folding states are recorded. Next, the applied constant force isreduced and the folding states accessible at the new force are recorded, and so on. Red traces obtained
at 15 pN of tension, yellow traces at ~12 pN, and blue traces at ~10 pN. U: unfolded state; intermediate
states: I-1 to I-4; PN: partial native state; N: native fold. Based on different extents of base-pairing ineach structural fold, each folding state is characterized by a distinct tether end-to-end distance that isexpressed in nm (dashed lines on y-axis in panel A and B, and on x-axis in panel C and D).
(A) Under 12.5 pN of clamping force (yellow), the 103-nt transcript transitions through intermediate
states I-2, I-3, and I-1, and eventually stably adopts state I-4 (i.e. cartoon 6 in Figure 2E). When theclamping force further drops to 10.5 pN (blue), the transcript undergoes rapidly transitions between twocompeting folds, I-4 and PN (cartoon 7 in Figure 2E).
(B) For the full-length SRP RNA, similar folding transitions as that of the nearly-completed transcript
are detected at clamping forces of 15 and ~12 pN, except that the 114-nt transcript can readily attain thenative long hairpin fold (N) at 12.8 pN (yellow trace). At 10.6 pN, the full-length transcript exclusively
adopts the native long hairpin fold.
(C-D) To construct the probability distributions of folded states visited by the SRP RNA as the clamping
force decreases (panel C for 103-nt transcript, and panel D for 114-nt transcript), we integrate eachforce-clamping trace along the time axis and align them by the RNA tether contour length. The
dominating population of native fold in panel D (dark blue peak) vs. the large population for theintermediate I-4 in panel C (dark blue peak), highlight that the last ~11 nt on the SRP RNA 3’-stretch
facilitates the attainment of native long hairpin fold.

A
C

B

D
F

E

Figure S4

Figure S4. Reduce P2 Pause in SRP RNA Transcription with Base-pairing
Compensating Mutations U82A/A25U; Related to Figure 3(A-B) Representative transcript extension traces with wild-type SRP RNA (WT) and A25U/U82A
mutant. Real-time transcription recorded at 8.6 pN (low force condition) with 50 μM NTP.
(C) Mean pause duration at P2 site for transcription of wild-type SRP RNA (n=12) and A25U/U82A
mutant (n=12), both recorded at low force condition ([NTP] = 50 μM).
(D-E) Representative transcript extension traces with wild-type SRP RNA (WT) and A25U/U82A
mutant. Real-time transcription recorded at 22 pN (high force condition) and with 50 μM NTP.
(F) Mean pause duration at P2 site for transcription of wild-type SRP RNA (n=15) and A25U/U82A
mutant (n=13), both recorded at high force condition ([NTP] = 50 μM).

A

C

rrnC
PLtetO-1

PLtetO-1
SRP
insertpZE21_SRP_rrnC2449 bp

KanR

B

ori

Modiﬁca(ons in plasmids harbored in surviving colonies (n=5 for each)
SRP RNA
variant

truncated/
impairedpromoter(1st PLtetO-1)

loss of
SRP insert

truncated/
impairedterminator(rrnC)

termina(onread-through(2nd PLtetO-1)

SRP_100









U18C_100









U32C/U34C_100









Figure S5

Figure S5. In Vivo Response toward SRP RNA Variant-carrying Plasmids; Related
to Figure 5(A) The plasmid construct of pZE21_SRP_rrnC, which is designed for precise transcription of SRP
RNA variants inserted therein (see Methods for plasmid sequence). PLtetO-1: a strong promoter (L forphage lambda), to which a tet operator is integrated within the promoter sequence so as to make it tightly
repressible. This operator-embodying promoter avoids excessive/unwanted nucleotide sequenceappended to the 5’-end of RNA product transcribed, which would be the case if a typical operatorappending promoter were used. rrnC: a strong terminator (Chen et al., 2013), immediately after the SRP
RNA. Then a second PLtetO-1 promoter located 54-nt downstream from the terminator. Binding of another
RNAP to this second promoter serves as a roadblock to ensure that the first RNAP properly terminates
on rrnC. KanR: kanamycin resistant. ori: high-copy-number colE1 origin of replication.
(B) Modifications of the above plasmid—such as impaired promoter, loss of SRP variant insert, and/or
inefficient termination—were found in colonies surviving from the otherwise lethal transcription of SRP
RNA truncated variants (Figure 5A, bottom half of the plate). Namely, these modifications compromise
the precise transcription of the 3’-end-truncated SRP RNA variants and alleviate the toxic impact oncell viability.
(C) Under stress condition—e.g. high salt (2x Luria broth, i.e. 50g/L) and stiff growth medium (2x agar,
i.e. 30g/L), as compared to normal LB agar plates (Figure 5), the difference in toxicity among plasmids
carrying different full-length SRP RNA variants is further accentuated. Specifically, a greater reduction
in surviving colonies is seen with the transformation of U18C_114 SRP RNA variant (lower right).
Here, it shows ~28% colony counts relative to that of WT_114, compared to ~44% relative to that of
WT_114 seen in Figure 5B.

Table S1. Persistence lengths of RNA and its conversion factors (nt/nm) under forces;
Related to STAR Methods (section: Unit conversion of transcription trajectories
recorded in nanometers into nucleotides)

Complete plasmid sequence of pZE21_wtSRP_FL_rrnC
Notations: ori (colE1) / KanR / PLtetO-1 / wtSRP_FL / rrnC / point mutation for U18C and U32C/U34C/ transcript truncation to 100-bp long

CTAGGCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA
CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTA
CCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG
TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGACTAGTGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAG
TTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAG
CGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCA
ATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGC
CATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGC
TTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCG
GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGG
CCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCG
GTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGA
GCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTT
TACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT
CGTCTTCACCTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGG

CCCCCACCCCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCGGATCCCATGGAGGCATTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCTTGCCATGTGTATGTGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCACATACTCTGATGATCCTTCGGGATCATTCATGGCAATCTAGACAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTAC