PMC 20201223 pmc.key 4820378 CC BY-NC no 0 0 10.1126/sciadv.1501397 4820378 27051866 1501397 e1501397 3 biomolecules nucleotides tRNA 3′-5′ addition reverse polymerization TLP crystal structure This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. surname:Kimura;given-names:Shoko surname:Suzuki;given-names:Tateki surname:Chen;given-names:Meirong surname:Kato;given-names:Koji surname:Yu;given-names:Jian surname:Nakamura;given-names:Akiyoshi surname:Tanaka;given-names:Isao surname:Yao;given-names:Min surname:Tanaka;given-names:Isao TITLE Keywords front 2 2016 0 Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein 0.99769545 protein_type cleaner0 2023-07-27T08:36:51Z MESH: Thg1-like protein ABSTRACT abstract 97 Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3′-5′) direction. 0.9979704 evidence cleaner0 2023-07-27T10:24:50Z DUMMY: Structures 0.99865925 protein_type cleaner0 2023-07-27T08:38:16Z MESH: Thg1-like proteins ABSTRACT abstract 232 Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. 0.9819144 protein_type cleaner0 2023-07-27T08:36:52Z MESH: Thg1-like protein 0.99417204 protein_type cleaner0 2023-07-27T08:37:27Z MESH: TLP 0.9968427 chemical cleaner0 2023-07-27T08:38:38Z CHEBI: transfer RNA 0.9986614 chemical cleaner0 2023-07-27T08:39:12Z CHEBI: tRNA 0.9991456 chemical cleaner0 2023-07-27T08:39:21Z CHEBI: adenosine 5′-triphosphate 0.99925095 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99916315 chemical cleaner0 2023-07-27T08:39:30Z CHEBI: guanosine 5′-triphosphate 0.99925977 chemical cleaner0 2023-07-27T08:39:34Z CHEBI: GTP 0.9986971 experimental_method cleaner0 2023-07-27T10:16:01Z MESH: Structural analyses 0.962661 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99868774 site cleaner0 2023-07-27T10:03:50Z SO: substrate binding sites site SO: cleaner0 2023-07-27T10:02:25Z reaction center 0.99890465 chemical cleaner0 2023-07-27T10:07:03Z CHEBI: Mg2+ site SO: cleaner0 2023-07-27T10:02:25Z reaction center 0.9965724 chemical cleaner0 2023-07-27T10:07:09Z CHEBI: nucleotide 0.9980839 protein_state cleaner0 2023-07-27T08:40:17Z DUMMY: bound to 0.99871296 site cleaner0 2023-07-27T10:05:45Z SO: second binding site bond_interaction MESH: cleaner0 2023-07-27T08:37:54Z Watson-Crick hydrogen bonds 0.8504399 chemical cleaner0 2023-07-27T09:32:05Z CHEBI: 5′-triphosphate 0.9985703 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.93671775 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.9894528 protein_type cleaner0 2023-07-27T10:06:42Z MESH: 3′-5′ elongation enzyme site SO: cleaner0 2023-07-27T10:05:27Z catalytic center 0.99867994 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99912554 protein cleaner0 2023-07-27T08:38:02Z PR: Thg1 0.99841225 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.65315086 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99869436 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9993358 protein cleaner0 2023-07-27T08:38:02Z PR: Thg1 protein_type MESH: cleaner0 2023-07-27T08:40:09Z tRNAHis-specific G−1 addition enzyme 0.9974462 chemical cleaner0 2023-07-27T08:40:24Z CHEBI: tRNAHis 0.99938846 protein cleaner0 2023-07-27T08:38:02Z PR: Thg1 0.9986511 oligomeric_state cleaner0 2023-07-27T08:39:42Z DUMMY: tetramer 0.9982622 structure_element cleaner0 2023-07-27T10:31:16Z SO: subunits 0.99881375 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9990613 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99875957 site cleaner0 2023-07-27T09:50:09Z SO: dimer interface structure_element SO: cleaner0 2023-07-27T10:22:09Z accepter stem 0.9910454 protein_state cleaner0 2023-07-27T10:29:14Z DUMMY: flexible 0.9992483 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.99854076 experimental_method cleaner0 2023-07-27T10:16:05Z MESH: mutational analyses 0.99759763 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.99900013 protein_state cleaner0 2023-07-27T08:40:16Z DUMMY: bound to 0.9980719 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99925965 protein cleaner0 2023-07-27T08:38:02Z PR: Thg1 0.99786204 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP INTRO title_1 1972 INTRODUCTION INTRO paragraph 1985 All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5′-3′ direction. This reaction involves the nucleophilic attack of a 3′-OH of the terminal nucleotide in the elongating chain on the α-phosphate of an incoming nucleotide. The energy in the high-energy bond of the incoming nucleotide is used for its addition [termed tail polymerization ]. This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. Thus, the energy in the triphosphate bond of the incoming nucleotide is not used for its own addition but is reserved for subsequent polymerization (that is, head polymerization) (Fig. 1). 0.9901839 chemical cleaner0 2023-07-27T10:07:14Z CHEBI: DNA 0.99599516 chemical cleaner0 2023-07-27T10:07:31Z CHEBI: RNA 0.9835027 chemical cleaner0 2023-07-27T09:08:06Z CHEBI: phosphate 0.9404228 chemical cleaner0 2023-07-27T10:07:15Z CHEBI: DNA 0.9858251 chemical cleaner0 2023-07-27T10:07:32Z CHEBI: RNA 0.99878734 chemical cleaner0 2023-07-27T09:07:59Z CHEBI: aminoacyl-tRNA 0.9985611 oligomeric_state cleaner0 2023-07-27T09:07:37Z DUMMY: monomer protein PR: cleaner0 2023-07-27T08:38:03Z Thg1 protein_type MESH: cleaner0 2023-07-27T08:36:52Z Thg1-like protein 0.99842346 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99820364 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99828124 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99920386 chemical cleaner0 2023-07-27T08:39:21Z CHEBI: adenosine 5′-triphosphate 0.9993376 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.9992138 chemical cleaner0 2023-07-27T08:39:30Z CHEBI: guanosine 5′-triphosphate 0.99931574 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.999152 chemical cleaner0 2023-07-27T09:08:33Z CHEBI: nucleoside 5′-triphosphate 0.9992663 chemical cleaner0 2023-07-27T09:08:37Z CHEBI: NTP 0.76323205 chemical cleaner0 2023-07-27T09:08:17Z CHEBI: pppN-tRNA chemical CHEBI: cleaner0 2023-07-27T09:24:41Z triphosphate 1501397-F1.jpg F1 FIG fig_title_caption 3237 Reaction schemes of 3′-5′ and 5′-3′ elongation. 1501397-F1.jpg F1 FIG fig_caption 3293 Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. Bottom: Reaction scheme of 5′-3′ elongation by DNA/RNA polymerases. In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. The energy of the incoming nucleotide is not used for its own addition but is reserved for the subsequent addition (head polymerization). In 5′-3′ elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization). protein PR: cleaner0 2023-07-27T08:38:03Z Thg1 protein_type MESH: cleaner0 2023-07-27T08:37:28Z TLP 0.99611807 protein_type cleaner0 2023-07-27T09:09:53Z MESH: DNA/RNA polymerases protein PR: cleaner0 2023-07-27T08:38:03Z Thg1 protein_type MESH: cleaner0 2023-07-27T08:37:28Z TLP chemical CHEBI: cleaner0 2023-07-27T10:08:10Z 5′-monophosphate 0.99824333 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9993061 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.9993092 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.996169 protein_type cleaner0 2023-07-27T09:09:53Z MESH: DNA/RNA polymerases INTRO paragraph 3878 The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes. However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. This finding suggests that TLPs may have potential functions other than tRNAHis maturation. TLPs have been shown to catalyze 5′-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template–dependent manner. This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. These observations suggest that TLPs may have more general functions in DNA/RNA repair. 0.99877626 taxonomy_domain cleaner0 2023-07-27T09:10:13Z DUMMY: eukaryotic 0.9991749 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99857557 protein_type cleaner0 2023-07-27T09:10:07Z MESH: tRNAHis guanylyltransferase 0.97453237 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.51417506 chemical cleaner0 2023-07-27T09:52:51Z CHEBI: guanosine 0.97779673 residue_number cleaner0 2023-07-27T10:14:27Z DUMMY: −1 0.93238014 residue_name_number cleaner0 2023-07-27T09:53:10Z DUMMY: G−1 0.96735996 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.99853724 protein_type cleaner0 2023-07-27T09:10:27Z MESH: histidyl-tRNA synthase 0.9990308 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9986656 taxonomy_domain cleaner0 2023-07-27T09:12:12Z DUMMY: eukaryotes 0.9842792 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9990934 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs 0.42650872 residue_name_number cleaner0 2023-07-27T09:53:21Z DUMMY: G−1 0.9991235 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs 0.9778497 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9990957 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs 0.99528116 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9990841 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs 0.98222923 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9904108 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.99877506 taxonomy_domain cleaner0 2023-07-27T09:12:04Z DUMMY: yeast 0.99906677 protein cleaner0 2023-07-27T10:14:00Z PR: Thg1p chemical CHEBI: cleaner0 2023-07-27T10:07:15Z DNA 0.99885345 taxonomy_domain cleaner0 2023-07-27T09:11:59Z DUMMY: plant 0.9990459 protein cleaner0 2023-07-27T10:14:08Z PR: ICA1 chemical CHEBI: cleaner0 2023-07-27T10:07:15Z DNA 0.99915147 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs chemical CHEBI: cleaner0 2023-07-27T10:07:15Z DNA chemical CHEBI: cleaner0 2023-07-27T10:07:32Z RNA INTRO paragraph 5092 The 3′-5′ addition reaction catalyzed by Thg1 occurs through three reaction steps. In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. In the second step, the 3′-OH of the incoming GTP attacks the activated intermediate, yielding pppG−1-tRNAHis. Finally, the pyrophosphate is removed, and mature pG−1-tRNAHis is created. The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. Thus, a detailed reaction mechanism remains unknown. 0.9993753 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9984634 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9819302 protein_type cleaner0 2023-07-27T09:54:03Z MESH: ribonuclease P chemical CHEBI: cleaner0 2023-07-27T09:54:24Z pre-tRNAHis 0.9991885 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99659437 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.99922943 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP chemical CHEBI: cleaner0 2023-07-27T09:54:56Z pppG−1-tRNAHis 0.9992478 chemical cleaner0 2023-07-27T09:55:05Z CHEBI: pyrophosphate 0.99007195 chemical cleaner0 2023-07-27T09:55:25Z CHEBI: pG−1-tRNAHis 0.99864215 evidence cleaner0 2023-07-27T10:24:58Z DUMMY: crystal structure 0.99866784 species cleaner0 2023-07-27T09:11:53Z MESH: human 0.99936336 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9993332 protein cleaner0 2023-07-27T09:11:18Z PR: HsThg1 0.93159294 site cleaner0 2023-07-27T10:02:20Z SO: catalytic core 0.99825877 protein_type cleaner0 2023-07-27T10:06:46Z MESH: 5′-3′ nucleotide polymerases protein_type MESH: cleaner0 2023-07-27T10:06:22Z T7 DNA/RNA polymerases 0.99781764 protein_type cleaner0 2023-07-27T09:11:09Z MESH: 3′-5′ elongation enzymes 0.99836665 protein_type cleaner0 2023-07-27T09:11:02Z MESH: 5′-3′ polymerases 0.998312 protein_type cleaner0 2023-07-27T09:11:02Z MESH: 5′-3′ polymerases 0.99878764 evidence cleaner0 2023-07-27T10:25:01Z DUMMY: crystal structure 0.48748377 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9984957 species cleaner0 2023-07-27T09:11:46Z MESH: Bacillus thuringiensis 0.9980994 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.9979635 site cleaner0 2023-07-27T10:08:44Z SO: active-site 0.9993067 protein cleaner0 2023-07-27T09:11:19Z PR: HsThg1 0.9980274 evidence cleaner0 2023-07-27T10:25:03Z DUMMY: structure 0.99843144 species cleaner0 2023-07-27T09:11:35Z MESH: Candida albicans 0.9993672 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99935013 protein cleaner0 2023-07-27T09:11:40Z PR: CaThg1 0.99900305 protein_state cleaner0 2023-07-27T09:15:20Z DUMMY: complexed with 0.9984999 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9954639 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99536276 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.998207 protein_type cleaner0 2023-07-27T10:06:51Z MESH: DNA/RNA polymerase 0.99610376 experimental_method cleaner0 2023-07-27T10:16:10Z MESH: structural analysis 0.99906045 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.99919957 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.998389 protein_state cleaner0 2023-07-27T10:29:29Z DUMMY: not bound INTRO paragraph 6452 Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. On the basis of these structures, we discuss the reaction mechanism of template-dependent reverse (3′-5′) polymerization in comparison with canonical 5′-3′ polymerization. 0.9828011 experimental_method cleaner0 2023-07-27T10:16:16Z MESH: solved 0.9475209 evidence cleaner0 2023-07-27T10:25:08Z DUMMY: structure 0.96883136 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9628024 taxonomy_domain cleaner0 2023-07-27T09:13:32Z DUMMY: methanogenic archaeon 0.99863946 species cleaner0 2023-07-27T09:13:04Z MESH: Methanosarcina acetivorans 0.99919146 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP 0.99593526 protein_state cleaner0 2023-07-27T09:12:25Z DUMMY: in complex with 0.9728952 chemical cleaner0 2023-07-27T09:13:26Z CHEBI: ppptRNAPheΔ1 0.9950616 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9991167 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9980457 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.99848324 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.8676416 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9974669 evidence cleaner0 2023-07-27T10:25:11Z DUMMY: structure 0.99928975 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.9993855 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP bond_interaction MESH: cleaner0 2023-07-27T09:12:49Z Watson-Crick base pair 0.9993098 residue_name_number cleaner0 2023-07-27T09:17:23Z DUMMY: C72 0.99910235 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.997968 evidence cleaner0 2023-07-27T10:25:15Z DUMMY: structures RESULTS title_1 7130 RESULTS RESULTS title_2 7138 Anticodon-independent binding of ppptRNAPheΔ1 to MaTLP 0.9988223 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9930177 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP RESULTS paragraph 7197 Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”). experimental_method MESH: cleaner0 2023-07-27T10:16:49Z biochemical experiments 0.99851924 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9988738 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.30236262 residue_name_number cleaner0 2023-07-27T10:15:20Z DUMMY: G1 0.9096241 experimental_method cleaner0 2023-07-27T10:17:02Z MESH: deleted 0.9993913 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99921095 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9407621 evidence cleaner0 2023-07-27T10:25:26Z DUMMY: crystal 0.9993493 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP 0.99859333 protein_state cleaner0 2023-07-27T09:15:19Z DUMMY: complexed with 0.9969944 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.8221576 experimental_method cleaner0 2023-07-27T10:17:05Z MESH: solved 0.9791672 evidence cleaner0 2023-07-27T10:25:33Z DUMMY: structure 0.99778384 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9974795 evidence cleaner0 2023-07-27T10:25:57Z DUMMY: crystal 0.99883157 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9836731 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP structure_element SO: melaniev@ebi.ac.uk 2023-07-28T13:58:56Z A structure_element SO: melaniev@ebi.ac.uk 2023-07-28T13:58:56Z B 0.9989492 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9988199 oligomeric_state cleaner0 2023-07-27T09:14:45Z DUMMY: dimers 0.9564452 evidence cleaner0 2023-07-27T10:25:49Z DUMMY: crystal 0.998803 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.998838 oligomeric_state cleaner0 2023-07-27T09:14:46Z DUMMY: dimers 0.9986577 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.99766195 evidence cleaner0 2023-07-27T10:25:52Z DUMMY: structure 0.99917907 complex_assembly cleaner0 2023-07-27T09:15:08Z GO: TLP-tRNA complex_assembly GO: cleaner0 2023-07-27T09:14:11Z CaThg1-tRNA 0.9992673 structure_element cleaner0 2023-07-27T10:31:25Z SO: AB 0.9992729 structure_element cleaner0 2023-07-27T10:31:30Z SO: CD 0.99889463 oligomeric_state cleaner0 2023-07-27T09:14:46Z DUMMY: dimers 0.9985355 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.99947053 protein cleaner0 2023-07-27T09:11:41Z PR: CaThg1 0.9041023 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9974681 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9992331 structure_element cleaner0 2023-07-27T10:31:25Z SO: AB 0.99891055 oligomeric_state cleaner0 2023-07-27T09:14:50Z DUMMY: dimer 0.9992483 structure_element cleaner0 2023-07-27T10:31:30Z SO: CD 0.99889743 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9977823 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.9994438 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP 0.9987863 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99582255 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA structure_element SO: cleaner0 2023-07-27T10:22:09Z accepter stem 0.9753643 structure_element cleaner0 2023-07-27T10:23:13Z SO: elbow region 0.99939775 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP protein_type MESH: cleaner0 2023-07-27T10:17:42Z anticodon-independent repair enzyme 0.99917156 complex_assembly cleaner0 2023-07-27T09:14:38Z GO: MaTLP-tRNA 0.9994748 protein cleaner0 2023-07-27T09:11:41Z PR: CaThg1 residue_name_number DUMMY: cleaner0 2023-07-27T10:18:02Z G−1 0.8723751 residue_name cleaner0 2023-07-27T09:29:31Z SO: His 0.95955354 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 1501397-F2.jpg F2 FIG fig_title_caption 8563 Structure of the MaTLP complex with ppptRNAPheΔ1. 0.99634486 evidence cleaner0 2023-07-27T10:26:01Z DUMMY: Structure 0.92571753 protein cleaner0 2023-07-27T09:13:09Z PR: MaTLP 0.98812664 protein_state cleaner0 2023-07-27T09:16:07Z DUMMY: complex with 0.99362344 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 1501397-F2.jpg F2 FIG fig_caption 8617 Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). Right: Left figure rotated by 90o. The CD dimer is omitted for clarity. The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). Residues important for binding are depicted in stick form. The β-hairpin region of molecule B is shown in red. 0.99868494 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9433621 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9990138 protein_state cleaner0 2023-07-27T08:40:17Z DUMMY: bound to 0.9982882 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.9988288 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.99934846 structure_element cleaner0 2023-07-27T10:31:25Z SO: AB 0.99937457 structure_element cleaner0 2023-07-27T10:31:30Z SO: CD 0.9989034 oligomeric_state cleaner0 2023-07-27T09:14:46Z DUMMY: dimers 0.96084297 oligomeric_state cleaner0 2023-07-27T10:13:52Z DUMMY: dimerized 0.99878424 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.8921645 evidence cleaner0 2023-07-27T10:26:21Z DUMMY: structure 0.9987697 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9981351 oligomeric_state cleaner0 2023-07-27T09:14:46Z DUMMY: dimers 0.99936455 structure_element cleaner0 2023-07-27T10:31:30Z SO: CD 0.9989243 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.99393797 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9991868 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.91121864 structure_element cleaner0 2023-07-27T10:23:13Z SO: elbow region 0.99934506 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin RESULTS paragraph 9133 The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex. 0.9842645 structure_element cleaner0 2023-07-27T10:23:13Z SO: elbow region 0.998276 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9993427 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.9993599 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.99948394 residue_name_number cleaner0 2023-07-27T09:16:27Z DUMMY: R215 structure_element SO: cleaner0 2023-07-27T10:20:20Z β-hairpin 0.99937385 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.99709296 bond_interaction cleaner0 2023-07-27T09:19:48Z MESH: hydrogen-bonded chemical CHEBI: cleaner0 2023-07-27T09:08:07Z phosphate 0.99939704 residue_name_number cleaner0 2023-07-27T09:16:32Z DUMMY: U55 0.99942434 residue_name_number cleaner0 2023-07-27T09:16:36Z DUMMY: G57 0.9994429 residue_name_number cleaner0 2023-07-27T09:16:42Z DUMMY: S213 0.9968627 bond_interaction cleaner0 2023-07-27T09:19:48Z MESH: hydrogen-bonded chemical CHEBI: cleaner0 2023-07-27T09:08:07Z phosphate 0.9994149 residue_name_number cleaner0 2023-07-27T09:16:37Z DUMMY: G57 0.99904877 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA structure_element SO: cleaner0 2023-07-27T10:20:20Z β-hairpin 0.73507226 protein_state cleaner0 2023-07-27T10:26:27Z DUMMY: disordered 0.99873954 evidence cleaner0 2023-07-27T10:26:24Z DUMMY: crystal structure 0.99900657 complex_assembly cleaner0 2023-07-27T09:14:11Z GO: CaThg1-tRNA RESULTS paragraph 9571 The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity. 0.99863183 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9986902 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99942315 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.9985697 residue_name_number cleaner0 2023-07-27T09:17:01Z DUMMY: G2 0.99695307 bond_interaction cleaner0 2023-07-27T09:19:48Z MESH: hydrogen-bonded 0.999418 residue_name_number cleaner0 2023-07-27T09:17:06Z DUMMY: R136 0.98731846 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.999443 residue_name_number cleaner0 2023-07-27T09:17:10Z DUMMY: N137 0.99947983 residue_name_number cleaner0 2023-07-27T09:17:06Z DUMMY: R136 0.99682266 bond_interaction cleaner0 2023-07-27T09:19:48Z MESH: hydrogen-bonded 0.9994659 residue_name_number cleaner0 2023-07-27T09:17:23Z DUMMY: C72 0.99681926 chemical cleaner0 2023-07-27T09:24:41Z CHEBI: triphosphate 0.9985638 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA residue_range DUMMY: cleaner0 2023-07-27T09:17:52Z D21-K26 0.88514954 chemical cleaner0 2023-07-27T10:10:55Z CHEBI: phosphates 0.9750867 bond_interaction cleaner0 2023-07-27T09:19:44Z MESH: coordinated to 0.9988883 chemical cleaner0 2023-07-27T09:18:00Z CHEBI: Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:18:14Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:18:47Z Mg2+ 0.99945253 protein cleaner0 2023-07-27T09:11:41Z PR: CaThg1 0.9994295 protein cleaner0 2023-07-27T09:11:19Z PR: HsThg1 0.9982023 evidence cleaner0 2023-07-27T10:26:31Z DUMMY: structures bond_interaction MESH: cleaner0 2023-07-27T09:20:10Z coordinated by 0.99948525 residue_name_number cleaner0 2023-07-27T09:19:11Z DUMMY: D21 0.9995098 residue_name_number cleaner0 2023-07-27T09:19:15Z DUMMY: D69 0.9994955 residue_name_number cleaner0 2023-07-27T09:19:19Z DUMMY: G22 0.9975606 experimental_method cleaner0 2023-07-27T10:18:16Z MESH: Mutation 0.9994524 residue_name_number cleaner0 2023-07-27T09:19:24Z DUMMY: D29 0.9994642 residue_name_number cleaner0 2023-07-27T09:19:29Z DUMMY: D76 0.99943143 protein cleaner0 2023-07-27T09:11:19Z PR: HsThg1 0.9994531 residue_name_number cleaner0 2023-07-27T09:19:11Z DUMMY: D21 0.99944884 residue_name_number cleaner0 2023-07-27T09:19:15Z DUMMY: D69 0.99944633 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP residue_name_number DUMMY: cleaner0 2023-07-27T09:56:58Z G−1 RESULTS title_2 10461 Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPheΔ1 complex 0.99917847 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.998625 complex_assembly cleaner0 2023-07-27T09:20:21Z GO: MaTLP-ppptRNAPheΔ1 RESULTS paragraph 10544 Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. These features are consistent with the fact that this elongation reaction is template-dependent. The 5′-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP. Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs. 0.9968804 evidence cleaner0 2023-07-27T10:26:35Z DUMMY: structure 0.9987251 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.99861515 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.5469938 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9993401 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.99941456 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.998014 experimental_method cleaner0 2023-07-27T10:18:28Z MESH: soaking 0.99915266 complex_assembly cleaner0 2023-07-27T09:20:22Z GO: MaTLP-ppptRNAPheΔ1 0.97418684 evidence cleaner0 2023-07-27T10:26:38Z DUMMY: crystal 0.9993868 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.9970028 evidence cleaner0 2023-07-27T10:26:48Z DUMMY: structure 0.8970383 chemical cleaner0 2023-07-27T10:11:00Z CHEBI: guanine 0.9992805 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.9721004 bond_interaction cleaner0 2023-07-27T08:37:55Z MESH: Watson-Crick hydrogen bonds 0.99924314 residue_name_number cleaner0 2023-07-27T09:17:23Z DUMMY: C72 0.99525213 bond_interaction cleaner0 2023-07-27T09:21:16Z MESH: base-stacking interactions 0.9992003 residue_name_number cleaner0 2023-07-27T09:17:01Z DUMMY: G2 0.9982686 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9052167 chemical cleaner0 2023-07-27T10:11:03Z CHEBI: guanine 0.9985051 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99938846 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP chemical CHEBI: cleaner0 2023-07-27T09:32:05Z 5′-triphosphate 0.9993123 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.99930036 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99679154 chemical cleaner0 2023-07-27T09:24:41Z CHEBI: triphosphate 0.99933904 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP chemical CHEBI: cleaner0 2023-07-27T09:20:58Z Mg2+ 0.9886169 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.997259 evidence cleaner0 2023-07-27T10:26:51Z DUMMY: structure site SO: cleaner0 2023-07-27T10:02:25Z reaction center 1501397-F3.jpg F3 FIG fig_title_caption 11780 Structural change of the tRNA (ppptRNAPheΔ1). 0.99411327 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9977137 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 1501397-F3.jpg F3 FIG fig_caption 11830 Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well. 0.6159382 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.8077976 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9969453 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.99880004 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.9989936 chemical cleaner0 2023-07-27T09:21:33Z CHEBI: GDPNP 0.9961423 evidence cleaner0 2023-07-27T10:26:57Z DUMMY: Structure 0.9985405 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.9974503 evidence cleaner0 2023-07-27T10:26:59Z DUMMY: Structure 0.9985354 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.99718434 experimental_method cleaner0 2023-07-27T10:18:51Z MESH: Superposition 0.9865745 evidence cleaner0 2023-07-27T10:27:01Z DUMMY: structures 0.99856323 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99900997 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.99818677 experimental_method cleaner0 2023-07-27T10:18:55Z MESH: Superposition 0.99859303 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9986745 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.99921167 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.5339933 chemical cleaner0 2023-07-27T09:24:40Z CHEBI: triphosphate RESULTS paragraph 12306 The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur. 0.99445456 chemical cleaner0 2023-07-27T09:24:41Z CHEBI: triphosphate 0.9992803 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.99835026 site cleaner0 2023-07-27T10:28:07Z SO: interface structure_element SO: cleaner0 2023-07-27T09:21:53Z A structure_element SO: cleaner0 2023-07-27T09:22:01Z B 0.9993624 residue_name_number cleaner0 2023-07-27T09:21:42Z DUMMY: R19 structure_element SO: cleaner0 2023-07-27T09:22:09Z A 0.9993773 residue_name_number cleaner0 2023-07-27T09:22:50Z DUMMY: R83 structure_element SO: cleaner0 2023-07-27T09:22:18Z B 0.9993542 residue_name_number cleaner0 2023-07-27T09:22:45Z DUMMY: K86 structure_element SO: cleaner0 2023-07-27T09:22:26Z B 0.9993461 residue_name_number cleaner0 2023-07-27T09:22:55Z DUMMY: R114 structure_element SO: cleaner0 2023-07-27T09:22:41Z A 0.99890375 protein_state cleaner0 2023-07-27T10:29:45Z DUMMY: well conserved 0.9977977 experimental_method cleaner0 2023-07-27T10:19:04Z MESH: mutation 0.99941695 protein cleaner0 2023-07-27T10:14:16Z PR: ScThg1 0.99936813 residue_name_number cleaner0 2023-07-27T09:23:00Z DUMMY: R27 0.9993944 residue_name_number cleaner0 2023-07-27T09:23:04Z DUMMY: R93 0.9993637 residue_name_number cleaner0 2023-07-27T09:23:09Z DUMMY: K96 0.99935395 residue_name_number cleaner0 2023-07-27T09:23:14Z DUMMY: R133 0.9931157 residue_name_number cleaner0 2023-07-27T10:11:15Z DUMMY: G−1 0.99587363 chemical cleaner0 2023-07-27T09:24:41Z CHEBI: triphosphate 0.9992716 chemical cleaner0 2023-07-27T09:21:34Z CHEBI: GDPNP 0.9977677 chemical cleaner0 2023-07-27T10:11:19Z CHEBI: Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:23:34Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:24:00Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:24:23Z Mg2+ bond_interaction MESH: cleaner0 2023-07-27T09:42:41Z coordinated by 0.99687046 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP chemical CHEBI: cleaner0 2023-07-27T09:24:41Z triphosphate 0.99195087 site cleaner0 2023-07-27T10:28:12Z SO: second nucleotide binding site 0.99942905 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9525255 site cleaner0 2023-07-27T10:28:15Z SO: second site 0.90256834 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate RESULTS title_2 13175 tRNA binding and repair experiments of the β-hairpin mutants experimental_method MESH: cleaner0 2023-07-27T10:19:35Z tRNA binding and repair experiments 0.99890774 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.9695342 protein_state cleaner0 2023-07-27T10:29:48Z DUMMY: mutants RESULTS paragraph 13239 To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. Then, tRNA binding and enzymatic activities were measured. β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). Experiments using the tRNAHisΔ1 substrate gave similar results (Fig. 4A). All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition. chemical CHEBI: cleaner0 2023-07-27T08:39:13Z tRNA 0.99931544 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin experimental_method MESH: cleaner0 2023-07-27T10:20:01Z created mutation variants 0.9993222 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin experimental_method MESH: cleaner0 2023-07-27T10:20:48Z tRNA binding and enzymatic activities were measured 0.96807307 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-Hairpin 0.8194407 protein_state cleaner0 2023-07-27T10:29:51Z DUMMY: deletion variant 0.9983492 mutant cleaner0 2023-07-27T09:24:58Z MESH: delR198-R215 0.9296625 chemical cleaner0 2023-07-27T09:25:56Z CHEBI: tRNAPheΔ1 0.99831027 mutant cleaner0 2023-07-27T09:24:59Z MESH: delR198-R215 0.99695975 mutant cleaner0 2023-07-27T09:25:09Z MESH: delG202-E210 0.99902546 protein_state cleaner0 2023-07-27T09:26:30Z DUMMY: wild type 0.98946804 experimental_method cleaner0 2023-07-27T10:21:34Z MESH: mutations 0.99896884 mutant cleaner0 2023-07-27T09:25:13Z MESH: N179A 0.99896014 mutant cleaner0 2023-07-27T09:25:17Z MESH: F174A 0.9989961 mutant cleaner0 2023-07-27T09:25:14Z MESH: N179A 0.99903995 mutant cleaner0 2023-07-27T09:25:26Z MESH: R188A 0.99906284 site cleaner0 2023-07-27T10:28:19Z SO: anticodon recognition site 0.99927264 complex_assembly cleaner0 2023-07-27T09:25:39Z GO: Thg1-tRNAHis 0.5787097 evidence cleaner0 2023-07-27T10:27:07Z DUMMY: structure 0.97306955 chemical cleaner0 2023-07-27T10:11:23Z CHEBI: tRNAHisΔ1 0.9987498 evidence cleaner0 2023-07-27T10:27:09Z DUMMY: crystal structure 0.99930507 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.84993595 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9993302 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.9987729 protein_state cleaner0 2023-07-27T10:29:54Z DUMMY: not well conserved 0.9993625 residue_name_number cleaner0 2023-07-27T09:16:27Z DUMMY: R215 0.8830756 protein_state cleaner0 2023-07-27T10:29:59Z DUMMY: Mutants 0.9989693 mutant cleaner0 2023-07-27T09:26:15Z MESH: R215A 0.9989299 mutant cleaner0 2023-07-27T09:26:16Z MESH: R215A 0.9990251 mutant cleaner0 2023-07-27T09:26:23Z MESH: S213A 0.9988954 protein_state cleaner0 2023-07-27T10:30:03Z DUMMY: completely conserved 0.99940014 residue_name_number cleaner0 2023-07-27T09:16:27Z DUMMY: R215 0.51008594 experimental_method cleaner0 2023-07-27T10:21:40Z MESH: changed 0.9953343 residue_name cleaner0 2023-07-27T09:58:38Z SO: alanine 0.99303716 protein_state cleaner0 2023-07-27T10:30:06Z DUMMY: conserved 0.99940646 residue_name_number cleaner0 2023-07-27T09:16:27Z DUMMY: R215 bond_interaction MESH: cleaner0 2023-07-27T09:58:17Z van der Waals contacts 0.99931306 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin chemical CHEBI: cleaner0 2023-07-27T08:39:13Z tRNA 1501397-F4.jpg F4 FIG fig_title_caption 14513 Mutational analysis of the β-hairpin and anticodon binding region. 0.9984823 experimental_method cleaner0 2023-07-27T10:21:45Z MESH: Mutational analysis 0.9993003 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.9987523 site cleaner0 2023-07-27T10:28:24Z SO: anticodon binding region 1501397-F4.jpg F4 FIG fig_caption 14583 The rates of guanylylation by various mutants were measured. Error bars represent the SD of three independent experiments. (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1. 0.99230766 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9945064 chemical cleaner0 2023-07-27T09:26:49Z CHEBI: ppptRNAHisΔ1 0.96006143 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.75529 protein_state cleaner0 2023-07-27T10:30:10Z DUMMY: mutants 0.9910155 chemical cleaner0 2023-07-27T10:09:04Z CHEBI: [α-32P]GTP 0.9991336 protein_state cleaner0 2023-07-27T09:27:36Z DUMMY: wild-type 0.53344727 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.9951326 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 0.9966877 chemical cleaner0 2023-07-27T09:27:17Z CHEBI: tRNAPheΔ1 0.99821585 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.9968773 chemical cleaner0 2023-07-27T09:27:29Z CHEBI: tRNAHisΔ−1 0.9707573 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.8338082 protein_state cleaner0 2023-07-27T10:30:12Z DUMMY: mutants 0.6254101 chemical cleaner0 2023-07-27T09:27:17Z CHEBI: tRNAPheΔ1 0.9869407 chemical cleaner0 2023-07-27T09:13:27Z CHEBI: ppptRNAPheΔ1 RESULTS title_2 15033 Termination of the elongation reaction by measuring the accepter stem structure_element SO: cleaner0 2023-07-27T10:22:09Z accepter stem RESULTS paragraph 15103 TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). How TLP distinguishes between tRNAs that need 5′-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown. The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. When the 5′-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length. The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7). This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates. 0.9963889 protein_type cleaner0 2023-07-27T09:10:32Z MESH: TLPs 0.9868966 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.95910984 residue_name_number cleaner0 2023-07-27T10:15:25Z DUMMY: G1 0.99755114 chemical cleaner0 2023-07-27T09:27:17Z CHEBI: tRNAPheΔ1 0.96142435 residue_name_number cleaner0 2023-07-27T10:15:29Z DUMMY: G1 0.9508771 residue_name_number cleaner0 2023-07-27T09:17:01Z DUMMY: G2 0.99773103 chemical cleaner0 2023-07-27T09:28:04Z CHEBI: tRNAPheΔ1,2 0.9463474 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.98515874 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99676067 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.99747026 evidence cleaner0 2023-07-27T10:27:15Z DUMMY: structure 0.9989264 complex_assembly cleaner0 2023-07-27T09:20:22Z GO: MaTLP-ppptRNAPheΔ1 0.9987936 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.9641728 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.9986859 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9985221 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.5924758 structure_element cleaner0 2023-07-27T10:23:12Z SO: elbow region 0.999376 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.80603194 structure_element cleaner0 2023-07-27T10:31:36Z SO: B 0.90232784 structure_element cleaner0 2023-07-27T10:31:40Z SO: A 0.9393595 protein_state cleaner0 2023-07-27T10:30:18Z DUMMY: flexible 0.99939483 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.99264103 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.86848617 structure_element cleaner0 2023-07-27T10:22:08Z SO: accepter stem experimental_method MESH: cleaner0 2023-07-27T10:24:01Z used computer graphics to examine 0.9993806 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.99490696 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.7337481 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem site SO: cleaner0 2023-07-27T10:23:06Z reaction site 0.9975822 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA structure_element SO: cleaner0 2023-07-27T10:22:09Z accepter stem 0.9953381 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9955665 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9954334 evidence cleaner0 2023-07-27T10:27:17Z DUMMY: structure 0.9975693 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.967975 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.99937016 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.77153134 structure_element cleaner0 2023-07-27T10:23:13Z SO: elbow region 0.9978011 chemical cleaner0 2023-07-27T09:27:17Z CHEBI: tRNAPheΔ1 0.9983471 chemical cleaner0 2023-07-27T09:28:10Z CHEBI: tRNAPheΔ1,2 0.99938065 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.99614185 evidence cleaner0 2023-07-27T10:27:19Z DUMMY: structures 0.9082034 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.98609275 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9883173 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA RESULTS title_2 16831 Dual binding mode for tRNA repair 0.99911565 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA RESULTS paragraph 16865 The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. This difference in the tRNA binding modes is closely related to their enzymatic functions. The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5′-truncated general tRNAs. This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin. 0.99838936 experimental_method cleaner0 2023-07-27T10:24:07Z MESH: structural analysis 0.99898773 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99926084 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99870765 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.9988476 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.99926776 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99909174 protein_state cleaner0 2023-07-27T08:40:17Z DUMMY: bound to 0.996358 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.99887127 oligomeric_state cleaner0 2023-07-27T08:39:42Z DUMMY: tetramer 0.999076 protein_type cleaner0 2023-07-27T08:37:28Z MESH: TLP 0.99910367 protein_state cleaner0 2023-07-27T08:40:17Z DUMMY: bound to 0.9971238 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.99889284 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer chemical CHEBI: cleaner0 2023-07-27T08:39:13Z tRNA protein_type MESH: cleaner0 2023-07-27T08:40:09Z tRNAHis-specific G−1 addition enzyme 0.99932766 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.99702764 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.59451216 structure_element cleaner0 2023-07-27T10:32:03Z SO: anticodon 0.9981139 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9986755 oligomeric_state cleaner0 2023-07-27T08:39:47Z DUMMY: tetrameric 0.99936885 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.998466 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.99264705 structure_element cleaner0 2023-07-27T10:31:25Z SO: AB 0.99889743 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9975226 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.99948126 structure_element cleaner0 2023-07-27T10:31:30Z SO: CD 0.99879384 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9991611 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9989042 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.9978206 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9984469 protein_state cleaner0 2023-07-27T10:30:26Z DUMMY: flexible 0.99937433 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin RESULTS paragraph 17811 Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. TLP has been shown to confer such catalytic activity on tRNAHisΔ−1 (Fig. 4B). Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. By having two different binding modes, TLP can manage this special feature of tRNAHis. 0.89148074 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9935821 chemical cleaner0 2023-07-27T10:11:27Z CHEBI: guanosine 0.9969999 residue_name_number cleaner0 2023-07-27T10:11:38Z DUMMY: G−1 0.99664366 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.8903893 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9946516 chemical cleaner0 2023-07-27T09:27:30Z CHEBI: tRNAHisΔ−1 0.93582135 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.4843938 protein_state cleaner0 2023-07-27T10:30:30Z DUMMY: mutants 0.9993315 structure_element cleaner0 2023-07-27T10:20:20Z SO: β-hairpin 0.98948985 protein_state cleaner0 2023-07-27T10:30:35Z DUMMY: truncated chemical CHEBI: cleaner0 2023-07-27T09:27:17Z tRNAPheΔ1 0.99827754 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.9803373 chemical cleaner0 2023-07-27T09:29:20Z CHEBI: GUG 0.9556936 residue_name cleaner0 2023-07-27T09:29:30Z SO: His 0.99863344 chemical cleaner0 2023-07-27T09:27:24Z CHEBI: tRNAPhe 0.99575424 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9732659 residue_name cleaner0 2023-07-27T09:29:31Z SO: His 0.9255783 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.60005647 protein_state cleaner0 2023-07-27T10:30:38Z DUMMY: variant 0.9991592 mutant cleaner0 2023-07-27T09:25:18Z MESH: F174A 0.99910396 mutant cleaner0 2023-07-27T09:25:14Z MESH: N179A 0.99916065 mutant cleaner0 2023-07-27T09:25:26Z MESH: R188A 0.9990769 site cleaner0 2023-07-27T10:28:28Z SO: anticodon recognition site 0.9992427 complex_assembly cleaner0 2023-07-27T09:25:39Z GO: Thg1-tRNAHis 0.8880766 evidence cleaner0 2023-07-27T10:27:28Z DUMMY: structure 0.99190134 chemical cleaner0 2023-07-27T09:27:30Z CHEBI: tRNAHisΔ−1 0.997844 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9966815 chemical cleaner0 2023-07-27T10:10:42Z CHEBI: tRNAs 0.95553994 residue_name cleaner0 2023-07-27T09:29:31Z SO: His 0.9989329 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 0.5086558 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.98881125 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP chemical CHEBI: cleaner0 2023-07-27T08:39:13Z tRNA 0.98896366 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.523069 structure_element cleaner0 2023-07-27T10:32:08Z SO: anticodon 0.7900665 residue_name cleaner0 2023-07-27T09:29:31Z SO: His 0.99240595 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.99671626 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9921144 residue_name_number cleaner0 2023-07-27T10:11:48Z DUMMY: G−1 0.99529797 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.8667127 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis DISCUSS title_1 19268 DISCUSSION DISCUSS paragraph 19279 The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. The reaction involves two steps. First, the 5′-phosphate is activated by GTP/ATP. Then, the activated phosphate is attacked by the incoming nucleotide, resulting in an extension by one nucleotide at the 5′-end. Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. On the basis of these structures, we will discuss the 3′-5′ addition reaction compared with canonical 5′-3′ elongation by DNA/RNA polymerases. protein PR: cleaner0 2023-07-27T08:38:03Z Thg1 protein_type MESH: cleaner0 2023-07-27T08:37:29Z TLP 0.9985511 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA chemical CHEBI: cleaner0 2023-07-27T09:35:20Z 5′-phosphate 0.9992725 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.999213 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99731773 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.98605746 experimental_method cleaner0 2023-07-27T10:24:29Z MESH: solved 0.9901758 evidence cleaner0 2023-07-27T10:27:31Z DUMMY: structures 0.9988562 protein cleaner0 2023-07-27T09:13:10Z PR: MaTLP 0.9983096 evidence cleaner0 2023-07-27T10:27:33Z DUMMY: structures 0.98613816 protein_type cleaner0 2023-07-27T09:09:54Z MESH: DNA/RNA polymerases DISCUSS paragraph 19866 Figure 5 is a schematic diagram of the 3′-5′ addition reaction of TLP. This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). Then, the elongation reaction of step 2 occurs (Fig. 5E). Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. Although these two reactions are similar in chemistry, their substrate characteristics are very different. It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center. 0.525246 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9989221 site cleaner0 2023-07-27T09:29:55Z SO: triphosphate binding sites 0.99723035 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.99826884 site cleaner0 2023-07-27T10:28:34Z SO: binding sites 0.99893254 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.9986119 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99674594 protein_state cleaner0 2023-07-27T08:40:17Z DUMMY: bound to 0.99489546 site cleaner0 2023-07-27T09:38:12Z SO: site 1 chemical CHEBI: cleaner0 2023-07-27T09:35:20Z 5′-phosphate 0.9989851 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA chemical CHEBI: cleaner0 2023-07-27T09:31:14Z Mg2+ 0.99080896 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.99920815 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.99906045 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99654704 evidence cleaner0 2023-07-27T10:27:37Z DUMMY: structure 0.9989104 complex_assembly cleaner0 2023-07-27T09:20:22Z GO: MaTLP-ppptRNAPheΔ1 0.6651541 chemical cleaner0 2023-07-27T10:10:56Z CHEBI: phosphates 0.9484136 bond_interaction cleaner0 2023-07-27T09:30:12Z MESH: coordinate with chemical CHEBI: cleaner0 2023-07-27T09:30:29Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:30:51Z Mg2+ 0.9949398 chemical cleaner0 2023-07-27T10:12:09Z CHEBI: nucleotide 0.9928668 site cleaner0 2023-07-27T09:38:06Z SO: site 2 bond_interaction MESH: cleaner0 2023-07-27T09:12:50Z Watson-Crick base pair 0.98880345 chemical cleaner0 2023-07-27T10:12:18Z CHEBI: nucleotide 0.9964449 chemical cleaner0 2023-07-27T10:12:23Z CHEBI: nucleotide chemical CHEBI: cleaner0 2023-07-27T09:31:45Z Mg2+ 0.9326544 chemical cleaner0 2023-07-27T09:32:04Z CHEBI: 5′-triphosphate 0.9990356 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9961376 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.9960509 evidence cleaner0 2023-07-27T10:27:40Z DUMMY: structure protein_type MESH: cleaner0 2023-07-27T09:32:31Z 3′-5′ elongation enzyme 0.99522007 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.9950315 protein_type cleaner0 2023-07-27T09:32:15Z MESH: 5′-3′ elongation enzymes 0.7864525 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9921572 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 1501397-F5.jpg F5 FIG fig_title_caption 21310 Schematic representation of the 3′-5′ elongation mechanism. 1501397-F5.jpg F5 FIG fig_caption 21374 (A) The reaction center overlapped with two triphosphate binding sites. A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). (C′) Structure before the elongation reaction (corresponding to Fig. 3A). The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). (D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B). The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction. site SO: cleaner0 2023-07-27T10:02:25Z reaction center 0.9989655 site cleaner0 2023-07-27T09:29:56Z SO: triphosphate binding sites 0.99841654 site cleaner0 2023-07-27T10:28:39Z SO: binding sites chemical CHEBI: cleaner0 2023-07-27T09:38:53Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:39:06Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:39:21Z Mg2+ 0.6204343 site cleaner0 2023-07-27T10:28:53Z SO: P 0.99869686 site cleaner0 2023-07-27T09:33:28Z SO: phosphate binding sites 0.9984143 site cleaner0 2023-07-27T10:29:02Z SO: binding site 0.77258825 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.99759597 chemical cleaner0 2023-07-27T08:39:13Z CHEBI: tRNA 0.9947387 chemical cleaner0 2023-07-27T10:12:28Z CHEBI: Mg2+ 0.9144296 evidence cleaner0 2023-07-27T10:27:45Z DUMMY: Structure 0.93158305 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.99546534 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.97349215 site cleaner0 2023-07-27T09:33:56Z SO: triphosphate binding site 1 chemical CHEBI: cleaner0 2023-07-27T09:34:45Z 5′-phosphate 0.9780151 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.998922 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.99861634 chemical cleaner0 2023-07-27T08:39:26Z CHEBI: ATP 0.99910563 chemical cleaner0 2023-07-27T09:34:16Z CHEBI: PPi 0.94754815 chemical cleaner0 2023-07-27T09:34:19Z CHEBI: inorganic pyrophosphate 0.96327895 evidence cleaner0 2023-07-27T10:27:48Z DUMMY: structure 0.9909759 evidence cleaner0 2023-07-27T10:27:50Z DUMMY: structure 0.98339015 evidence cleaner0 2023-07-27T10:27:53Z DUMMY: Structure 0.8053533 chemical cleaner0 2023-07-27T09:32:05Z CHEBI: 5′-triphosphate 0.99874806 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.9983541 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.9921504 evidence cleaner0 2023-07-27T10:27:55Z DUMMY: Structure 0.9979594 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP 0.993615 bond_interaction cleaner0 2023-07-27T10:00:48Z MESH: Watson-Crick hydrogen bond 0.91873455 chemical cleaner0 2023-07-27T10:12:49Z CHEBI: nucleotide 0.97934216 residue_number cleaner0 2023-07-27T10:15:14Z DUMMY: 72 0.9953772 bond_interaction cleaner0 2023-07-27T10:00:56Z MESH: base-stacking interaction 0.98064464 residue_name_number cleaner0 2023-07-27T09:17:02Z DUMMY: G2 0.934088 chemical cleaner0 2023-07-27T09:32:05Z CHEBI: 5′-triphosphate 0.9988329 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.99860734 chemical cleaner0 2023-07-27T08:39:35Z CHEBI: GTP chemical CHEBI: cleaner0 2023-07-27T10:13:05Z Mg2+ 0.946844 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.99588984 chemical cleaner0 2023-07-27T09:24:41Z CHEBI: triphosphate 0.9973099 chemical cleaner0 2023-07-27T10:13:28Z CHEBI: nucleotide 0.9555763 site cleaner0 2023-07-27T09:38:12Z SO: site 1 DISCUSS paragraph 22881 Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. In these reactions, the roles of the two Mg ions are identical. Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core. 0.9598534 site cleaner0 2023-07-27T10:02:25Z SO: reaction center chemical CHEBI: cleaner0 2023-07-27T09:40:35Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:40:50Z Mg2+ 0.9991924 protein_state cleaner0 2023-07-27T10:01:21Z DUMMY: conserved 0.8309485 site cleaner0 2023-07-27T10:02:20Z SO: catalytic core 0.99529254 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9892129 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.99833184 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA protein PR: cleaner0 2023-07-27T09:41:57Z T7 RNA polymerase protein_type MESH: cleaner0 2023-07-27T09:40:04Z 5′-3′ DNA/RNA polymerase 0.9950848 chemical cleaner0 2023-07-27T10:07:32Z CHEBI: RNA 0.992826 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.9984529 chemical cleaner0 2023-07-27T10:13:31Z CHEBI: Mg chemical CHEBI: cleaner0 2023-07-27T09:40:20Z Mg2+ 0.99487984 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9904358 chemical cleaner0 2023-07-27T10:07:32Z CHEBI: RNA protein PR: cleaner0 2023-07-27T09:41:58Z T7 RNA polymerase chemical CHEBI: cleaner0 2023-07-27T09:41:06Z Mg2+ chemical CHEBI: cleaner0 2023-07-27T09:32:05Z 5′-triphosphate 0.9985898 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.9954899 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP protein PR: cleaner0 2023-07-27T09:41:58Z T7 RNA polymerase 0.9950562 chemical cleaner0 2023-07-27T10:13:38Z CHEBI: Mg2+ bond_interaction MESH: cleaner0 2023-07-27T09:42:41Z coordinated by 0.9991054 protein_state cleaner0 2023-07-27T10:01:23Z DUMMY: conserved 0.9874692 residue_name cleaner0 2023-07-27T09:42:20Z SO: Asp 0.9995602 residue_name_number cleaner0 2023-07-27T09:19:11Z DUMMY: D21 0.9995504 residue_name_number cleaner0 2023-07-27T09:19:15Z DUMMY: D69 0.9883781 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9991966 protein_state cleaner0 2023-07-27T10:01:25Z DUMMY: conserved 0.8672683 site cleaner0 2023-07-27T10:02:20Z SO: catalytic core 1501397-F6.jpg F6 FIG fig_title_caption 23926 Structures of template-dependent nucleotide elongation in the 3′-5′ and 5′-3′ directions. 0.99832505 evidence cleaner0 2023-07-27T10:28:01Z DUMMY: Structures 1501397-F6.jpg F6 FIG fig_caption 24024 Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). Red arrows represent elongation directions. In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. Green spheres represent Mg2+ ions. 0.9417723 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP protein PR: cleaner0 2023-07-27T09:41:58Z T7 RNA polymerase 0.9881513 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.99836165 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.99744284 chemical cleaner0 2023-07-27T10:07:32Z CHEBI: RNA 0.9937757 chemical cleaner0 2023-07-27T09:08:07Z CHEBI: phosphate 0.99891865 chemical cleaner0 2023-07-27T10:13:43Z CHEBI: Mg2+ DISCUSS paragraph 24622 Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6). In spite of this difference, their fundamental reaction scheme is conserved. However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP. For this reason, TLP requires a mechanism that activates the 5′-terminus of the tRNA during the initial step of the reaction. Our analysis showed that the initial activation and subsequent elongation reactions occur sequentially at one reaction center. In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center. TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed. 0.998965 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.9934815 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.9966459 protein_type cleaner0 2023-07-27T09:09:54Z MESH: DNA/RNA polymerases 0.8994305 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.88565826 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.99891484 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.86371475 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.99890584 site cleaner0 2023-07-27T10:03:49Z SO: substrate binding sites 0.9167209 site cleaner0 2023-07-27T10:02:25Z SO: reaction center 0.7830006 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.999042 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA bond_interaction MESH: cleaner0 2023-07-27T09:43:12Z Watson-Crick base pairing 0.5639086 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.99901265 site cleaner0 2023-07-27T10:03:43Z SO: activation reaction site 0.9269032 protein_type cleaner0 2023-07-27T10:06:57Z MESH: 3′-5′ elongation enzyme 0.98154014 protein_type cleaner0 2023-07-27T09:43:20Z MESH: 5′-3′ elongation enzyme 0.99903107 site cleaner0 2023-07-27T10:03:44Z SO: activation reaction site 0.94371027 protein_type cleaner0 2023-07-27T09:43:20Z MESH: 5′-3′ elongation enzyme DISCUSS paragraph 26101 Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. The active site of this enzyme is created at the dimerization interface. The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction. Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases. 0.586542 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP 0.9988463 site cleaner0 2023-07-27T10:05:33Z SO: catalytic center 0.99679774 protein_type cleaner0 2023-07-27T09:32:16Z MESH: 5′-3′ elongation enzymes 0.99908924 site cleaner0 2023-07-27T10:29:07Z SO: active site 0.99904776 site cleaner0 2023-07-27T10:03:33Z SO: dimerization interface 0.98878664 structure_element cleaner0 2023-07-27T10:22:09Z SO: accepter stem 0.9981711 chemical cleaner0 2023-07-27T08:39:14Z CHEBI: tRNA 0.98732823 residue_name_number cleaner0 2023-07-27T10:03:23Z DUMMY: G−1 0.9960594 chemical cleaner0 2023-07-27T08:40:25Z CHEBI: tRNAHis 0.9988643 oligomeric_state cleaner0 2023-07-27T09:14:51Z DUMMY: dimer 0.9988193 oligomeric_state cleaner0 2023-07-27T09:14:46Z DUMMY: dimers 0.9983082 experimental_method cleaner0 2023-07-27T10:24:40Z MESH: structural analysis 0.6366878 protein_type cleaner0 2023-07-27T08:37:29Z MESH: TLP protein_type MESH: cleaner0 2023-07-27T08:40:09Z tRNAHis-specific G−1 addition enzyme 0.99843353 protein cleaner0 2023-07-27T08:38:03Z PR: Thg1 protein_type MESH: cleaner0 2023-07-27T08:37:29Z TLP 0.9947258 protein_type cleaner0 2023-07-27T09:43:52Z MESH: template-dependent polymerases METHODS title_1 27219 MATERIALS AND METHODS METHODS title_2 27241 Plasmid construction METHODS paragraph 27262 Genomic DNA from M. acetivorans NBRC100939 was obtained from the NITE Biological Resource Center. The MaTLP gene was amplified by polymerase chain reaction from genomic DNA. The DNA fragment encoding MaTLP was then cloned between the Nde I and Xho I restriction sites in a pET26b vector with a C-terminal His tag. In the MaTLP gene, the amber stop codon (UAG) at position 142 was translated as Pyl. To express the full-length MaTLP in Escherichia coli, the TAG codon was altered to TGG (encoding Trp) with the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) as previously described. The inserted sequence was verified by DNA sequencing. METHODS title_2 27915 Preparation of MaTLP and mutants METHODS paragraph 27948 Plasmids were transformed into E. coli strain BL21 (DE3) pLysSRARE by electroporation, and cells were grown in LB medium containing kanamycin (25 μg/ml) and chloramphenicol (34 μg/ml) at 37°C until reaching an optical density at 600 nm (OD600) of 0.45. The cells were then induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 250 μM and shifted to 18°C for approximately 20 hours before harvest. The cells were harvested and resuspended in buffer A [50 mM Hepes-NaOH (pH 7.5), 1 M NaCl, 4 mM MgCl2, 10% glycerol, 0.5 mM β-mercaptoethanol, lysozyme (0.5 mg/ml), and deoxyibonuclease (0.1 mg/ml)]. After sonication and centrifugation, the His6-tagged protein was purified by immobilized metal-ion affinity chromatography using a HisTrap HP column (GE Healthcare). The sample was washed with 75 mM imidazole and eluted with a 75 to 400 mM imidazole gradient in buffer B [50 mM tris-HCl (pH 7.5), 500 mM NaCl, 4 mM MgCl2, 20% glycerol, and 0.5 mM β-mercaptoethanol]. Then, the collected fractions were diluted in 300 mM NaCl with buffer C [25 mM tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, and 1 mM dithiothreitol (DTT)] and further purified on a HiTrap Heparin HP column (GE Healthcare) by elution with a 300 to 1000 mM NaCl gradient in buffer C. Finally, the protein was loaded onto a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with buffer D [20 mM Hepes-NaOH (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. The protein was concentrated to 3.9 mg/ml by ultrafiltration. All MaTLP mutants were constructed with the QuikChange Site-Directed Mutagenesis Kit. MaTLP mutants were purified by a HisTrap HP column for RNA binding assay and further purified by a HiLoad 16/60 Superdex 200 prep grade column for 3′-5′ nucleotide addition assay. METHODS title_2 29779 Preparation of tRNA and its mutants METHODS paragraph 29815 tRNA transcripts derived from yeast tRNAPhe and tRNAHis were prepared using T7 RNA polymerase as previously described. ppptRNA transcripts were prepared by excluding guanosine 5′-monophosphate (GMP) from the reaction mixture. Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described. Pooled tRNAs were precipitated with isopropanol and dissolved in buffer E [20 mM Hepes-NaOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2]. chemical CHEBI: cleaner0 2023-07-27T10:10:42Z tRNAs chemical CHEBI: cleaner0 2023-07-27T10:10:42Z tRNAs METHODS title_2 30276 Preparation of the MaTLP-ppptRNAPheΔ1 complex METHODS paragraph 30326 MaTLP and ppptRNAPheΔ1 (tRNAPhe with a triphosphorylated 5′-end and deleted G1) were mixed in a molar ratio of 1.7:1 and incubated for 30 min at room temperature. The mixture was then loaded onto a HiLoad 16/60 Superdex 200 prep grade column equilibrated with buffer F [20 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. Fractions containing the MaTLP-ppptRNAPheΔ1 complex were mixed with 1 mM spermine and concentrated to an OD280 of 16 by ultrafiltration. METHODS title_2 30822 Crystallization and data collection METHODS paragraph 30858 All crystallization experiments were performed with the sitting-drop vapor diffusion method at 293 K. Initial crystals of MaTLP were obtained by mixing 1 μl of protein solution (3.9 mg/ml) with 1 μl of a reservoir solution containing 0.1 M Hepes-NaOH buffer (pH 7.5), 0.2 M magnesium chloride, and 30% polyethylene glycol 400 (PEG 400). MaTLP-GTP complex crystals were obtained by soaking the MaTLP crystals in the above reservoir solution supplemented with 1 mM GTP overnight. High-resolution crystals of MaTLP in apo form (MaTLP-apo) were obtained unexpectedly by mixing MaTLP with tRNAHis in 0.1 M sodium/potassium phosphate (pH 6.2) containing 2.5 M NaCl. Crystals of the MaTLP-ppptRNAPheΔ1 complex were obtained from a solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 37% PEG3350, and 10 mM praseodymium (III) acetate. Crystals of the MaTLP-ppptRNAPheΔ1-GDPNP complex were obtained by soaking MaTLP-ppptRNAPheΔ1 complex crystals in a reservoir solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 30% PEG3350, 5% glycerol, and 15 mM GDPNP overnight. Crystals of MaTLP-apo and MaTLP-GTP were cryoprotected with a reservoir solution containing 50% PEG400 before flash-cooling, whereas crystals of the MaTLP-ppptRNAPheΔ1-GDPNP and MaTLP-ppptRNAPheΔ1 complexes were flash-cooled without any cryoprotectant under a stream of liquid nitrogen at 100 K. X-ray diffraction data were collected from beamline BL41XU at SPring-8 (Hyogo, Japan) and beamlines BL5A and BL17A at Photon Factory (Ibaraki, Japan). All diffraction data were indexed, integrated, scaled, and merged using XDS. METHODS title_2 32495 Structure determination and refinement METHODS paragraph 32534 The crystal structure of MaTLP-apo was determined by the molecular replacement (MR) method with Molrep, using the protomer structure of CaThg1 (PDB ID: 3WBZ) as a search model. The protomer structure of MaTLP-apo was then used as a search model to solve the structures of MaTLP-GTP. The crystal structure of the MaTLP-ppptRNAPheΔ1 complex was determined by the MR method with PHASER, using the protomer structures of MaTLP-apo and tRNAPhe from Saccharomyces cerevisiae (PDB ID: 1EHZ) as search models. The structure of the MaTLP-ppptRNAPheΔ1 complex was then used as a search model to solve the MaTLP-ppptRNAPheΔ1-GDPNP complex structure. Initial protein models were fitted manually using Coot, and tRNA models were automatically rebuilt by LAFIRE_NAFIT; these models were then refined using phenix.refine. The data collection and refinement statistics are summarized in Table 1. All structure figures were generated by PyMol. T1.xml T1 TABLE table_title_caption 33473 Summary of data collection and refinement statistics. T1.xml T1 TABLE table_caption 33527 Values in parentheses are for the highest-resolution shell. PF, Photon Factory; Rmsd, root-mean-square deviation. T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><col width="%" span="1"/><thead><tr><td valign="top" align="left" scope="col" rowspan="1" colspan="1"/><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-apo</bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-GTP</bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-ppptRNA^PheΔ₁</bold></td><td valign="top" align="center" scope="col" rowspan="1" colspan="1"><bold><italic>Ma</italic>TLP-ppptRNA^PheΔ₁-GDPNP</bold></td></tr></thead><tbody><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">PDB ID</td><td valign="top" align="center" rowspan="1" colspan="1">5AXK</td><td valign="top" align="center" rowspan="1" colspan="1">5AXL</td><td valign="top" align="center" rowspan="1" colspan="1">5AXM</td><td valign="top" align="center" rowspan="1" colspan="1">5AXN</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1"><bold>Data collection</bold></td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Beamline</td><td valign="top" align="center" rowspan="1" colspan="1">SPring-8 BL41XU</td><td valign="top" align="center" rowspan="1" colspan="1">SPring-8 BL41XU</td><td valign="top" align="center" rowspan="1" colspan="1">PF BL17A</td><td valign="top" align="center" rowspan="1" colspan="1">PF BL5A</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Space group</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>C</italic>222₁</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>C</italic>222₁</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>I</italic>222</td><td valign="top" align="center" rowspan="1" colspan="1"><italic>I</italic>222</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Unit cell parameters <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">98.3, 120.5, 157.4</td><td valign="top" align="center" rowspan="1" colspan="1">103.1, 115.7, 144.9</td><td valign="top" align="center" rowspan="1" colspan="1">75.3, 127.6, 143.8</td><td valign="top" align="center" rowspan="1" colspan="1">82.3, 134.1, 147.4</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Wavelength (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.9780</td><td valign="top" align="center" rowspan="1" colspan="1">1.0000</td><td valign="top" align="center" rowspan="1" colspan="1">0.97319</td><td valign="top" align="center" rowspan="1" colspan="1">1.0000</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Resolution range (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.29 (2.43–2.29)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.99 (3.17–2.99)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.21 (2.34–2.21)</td><td valign="top" align="center" rowspan="1" colspan="1">50.0–2.70 (2.87–2.70)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  <italic>R</italic>_meas (%)*</td><td valign="top" align="center" rowspan="1" colspan="1">8.9 (76.3)</td><td valign="top" align="center" rowspan="1" colspan="1">15.2 (90.0)</td><td valign="top" align="center" rowspan="1" colspan="1">9.7 (74.4)</td><td valign="top" align="center" rowspan="1" colspan="1">11.0 (87.2)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  CC_1/2 (%)</td><td valign="top" align="center" rowspan="1" colspan="1">99.8 (80.4)</td><td valign="top" align="center" rowspan="1" colspan="1">99.5 (81.2)</td><td valign="top" align="center" rowspan="1" colspan="1">99.9 (83.6)</td><td valign="top" align="center" rowspan="1" colspan="1">99.9 (83.5)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  〈<italic>I</italic>/σ(<italic>I</italic>)〉</td><td valign="top" align="center" rowspan="1" colspan="1">14.7 (2.8)</td><td valign="top" align="center" rowspan="1" colspan="1">12.0 (2.6)</td><td valign="top" align="center" rowspan="1" colspan="1">19.4 (3.2)</td><td valign="top" align="center" rowspan="1" colspan="1">16.9 (2.5)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Completeness (%)</td><td valign="top" align="center" rowspan="1" colspan="1">98.3 (93.8)</td><td valign="top" align="center" rowspan="1" colspan="1">98.8 (93.4)</td><td valign="top" align="center" rowspan="1" colspan="1">99.7 (98.6)</td><td valign="top" align="center" rowspan="1" colspan="1">99.7 (99.3)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Redundancy</td><td valign="top" align="center" rowspan="1" colspan="1">6.7 (6.6)</td><td valign="top" align="center" rowspan="1" colspan="1">7.2 (7.2)</td><td valign="top" align="center" rowspan="1" colspan="1">7.4 (7.3)</td><td valign="top" align="center" rowspan="1" colspan="1">8.1 (8.2)</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1"><bold>Refinement</bold></td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  No. of reflections</td><td valign="top" align="center" rowspan="1" colspan="1">41,650</td><td valign="top" align="center" rowspan="1" colspan="1">17,581</td><td valign="top" align="center" rowspan="1" colspan="1">35,102</td><td valign="top" align="center" rowspan="1" colspan="1">22,669</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  <italic>R</italic>_work/<italic>R</italic>_free (%)^†</td><td valign="top" align="center" rowspan="1" colspan="1">20.6/24.0</td><td valign="top" align="center" rowspan="1" colspan="1">21.5/25.3</td><td valign="top" align="center" rowspan="1" colspan="1">21.6/24.3</td><td valign="top" align="center" rowspan="1" colspan="1">22.5/26.7</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  No. of atoms</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Macromolecules</td><td valign="top" align="center" rowspan="1" colspan="1">3760</td><td valign="top" align="center" rowspan="1" colspan="1">3622</td><td valign="top" align="center" rowspan="1" colspan="1">5247</td><td valign="top" align="center" rowspan="1" colspan="1">5142</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Ligand/ion</td><td valign="top" align="center" rowspan="1" colspan="1">30</td><td valign="top" align="center" rowspan="1" colspan="1">68</td><td valign="top" align="center" rowspan="1" colspan="1">36</td><td valign="top" align="center" rowspan="1" colspan="1">101</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Water</td><td valign="top" align="center" rowspan="1" colspan="1">89</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">102</td><td valign="top" align="center" rowspan="1" colspan="1">16</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  <italic>B</italic>-factors (Ų)</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Macromolecules</td><td valign="top" align="center" scope="col" rowspan="1" colspan="1">57.0</td><td valign="top" align="center" rowspan="1" colspan="1">68.4</td><td valign="top" align="center" rowspan="1" colspan="1">45.3</td><td valign="top" align="center" rowspan="1" colspan="1">57.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Ligand/ion</td><td valign="top" align="center" rowspan="1" colspan="1">60.5</td><td valign="top" align="center" rowspan="1" colspan="1">86.2</td><td valign="top" align="center" rowspan="1" colspan="1">46.6</td><td valign="top" align="center" rowspan="1" colspan="1">59.9</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Water</td><td valign="top" align="center" rowspan="1" colspan="1">49.0</td><td valign="top" align="center" rowspan="1" colspan="1">59.5</td><td valign="top" align="center" rowspan="1" colspan="1">33.0</td><td valign="top" align="center" rowspan="1" colspan="1">38.1</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">  Estimated coordinate error (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.32</td><td valign="top" align="center" rowspan="1" colspan="1">0.48</td><td valign="top" align="center" rowspan="1" colspan="1">0.25</td><td valign="top" align="center" rowspan="1" colspan="1">0.41</td></tr><tr><td colspan="5" valign="top" align="left" scope="row" rowspan="1">  Rmsd from ideal</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Bond lengths (Å)</td><td valign="top" align="center" rowspan="1" colspan="1">0.009</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td><td valign="top" align="center" rowspan="1" colspan="1">0.003</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">    Bond angles (°)</td><td valign="top" align="center" rowspan="1" colspan="1">1.11</td><td valign="top" align="center" rowspan="1" colspan="1">0.92</td><td valign="top" align="center" rowspan="1" colspan="1">0.72</td><td valign="top" align="center" rowspan="1" colspan="1">0.80</td></tr></tbody></table> 33641 MaTLP-apo MaTLP-GTP MaTLP-ppptRNAPheΔ1 MaTLP-ppptRNAPheΔ1-GDPNP PDB ID 5AXK 5AXL 5AXM 5AXN Data collection   Beamline SPring-8 BL41XU SPring-8 BL41XU PF BL17A PF BL5A   Space group C2221 C2221 I222 I222   Unit cell parameters a, b, c (Å) 98.3, 120.5, 157.4 103.1, 115.7, 144.9 75.3, 127.6, 143.8 82.3, 134.1, 147.4   Wavelength (Å) 0.9780 1.0000 0.97319 1.0000   Resolution range (Å) 50.0–2.29 (2.43–2.29) 50.0–2.99 (3.17–2.99) 50.0–2.21 (2.34–2.21) 50.0–2.70 (2.87–2.70)   Rmeas (%)* 8.9 (76.3) 15.2 (90.0) 9.7 (74.4) 11.0 (87.2)   CC1/2 (%) 99.8 (80.4) 99.5 (81.2) 99.9 (83.6) 99.9 (83.5)   〈I/σ(I)〉 14.7 (2.8) 12.0 (2.6) 19.4 (3.2) 16.9 (2.5)   Completeness (%) 98.3 (93.8) 98.8 (93.4) 99.7 (98.6) 99.7 (99.3)   Redundancy 6.7 (6.6) 7.2 (7.2) 7.4 (7.3) 8.1 (8.2) Refinement   No. of reflections 41,650 17,581 35,102 22,669   Rwork/Rfree (%)† 20.6/24.0 21.5/25.3 21.6/24.3 22.5/26.7   No. of atoms     Macromolecules 3760 3622 5247 5142     Ligand/ion 30 68 36 101     Water 89 8 102 16   B-factors (Å2)     Macromolecules 57.0 68.4 45.3 57.3     Ligand/ion 60.5 86.2 46.6 59.9     Water 49.0 59.5 33.0 38.1   Estimated coordinate error (Å) 0.32 0.48 0.25 0.41   Rmsd from ideal     Bond lengths (Å) 0.009 0.003 0.003 0.003     Bond angles (°) 1.11 0.92 0.72 0.80 T1.xml T1 TABLE table_foot 35061 *Rmeas = Σhkl {N(hkl)/[N(hkl) − 1]}1/2 Σi | Ii(hkl) − 〈I(hkl)〉 |/Σhkl Σi Ii(hkl), where 〈I(hkl)〉 and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively. T1.xml T1 TABLE table_foot 35279 †Rwork = Σhkl ||Fobs| − |Fcalc||/Σhkl |Fobs|; Rfree was calculated for 5% randomly selected test sets that were not used in the refinement. METHODS title_2 35427 Nucleotide addition assay METHODS paragraph 35453 Nucleotide addition assays were performed as previously described. A reaction mixture containing 25 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 10 mM MgCl2, 3 mM DTT, 5% glycerol, 0.1 μM [α-32P]GTP, 100 μM GTP, 1 μM MaTLP variants, and 10 μM tRNA transcript was incubated at 30°C for 2 hours. Then, the reaction was quenched with phenol/chloroform, and the supernatant was resolved on a 10% polyacrylamide gel containing 8 M urea. The radioactivity was visualized with a BAS-1800 II bioimaging analyzer (Fujifilm). METHODS title_2 35969 tRNA binding assay METHODS paragraph 35988 A reaction mixture containing 34 μM MaTLP variants and 20 μM tRNA transcript was incubated in buffer F at room temperature for 30 min. 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