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1.
Nucleic Acids Res ; 49(17): 10046-10060, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34417618

RESUMO

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.

2.
Elife ; 102021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34382933

RESUMO

N1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.

3.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
4.
Nat Commun ; 12(1): 328, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436566

RESUMO

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Bacteriano , RNA de Transferência/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilação , Anticódon/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Hidrólise , Metilação , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo
5.
Wiley Interdiscip Rev RNA ; 11(6): e1609, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32533808

RESUMO

A major threat to public health is the resistance and persistence of Gram-negative bacteria to multiple drugs during antibiotic treatment. The resistance is due to the ability of these bacteria to block antibiotics from permeating into and accumulating inside the cell, while the persistence is due to the ability of these bacteria to enter into a nonreplicating state that shuts down major metabolic pathways but remains active in drug efflux. Resistance and persistence are permitted by the unique cell envelope structure of Gram-negative bacteria, which consists of both an outer and an inner membrane (OM and IM, respectively) that lay above and below the cell wall. Unexpectedly, recent work reveals that m1 G37 methylation of tRNA, at the N1 of guanosine at position 37 on the 3'-side of the tRNA anticodon, controls biosynthesis of both membranes and determines the integrity of cell envelope structure, thus providing a novel link to the development of bacterial resistance and persistence to antibiotics. The impact of m1 G37-tRNA methylation on Gram-negative bacteria can reach further, by determining the ability of these bacteria to exit from the persistence state when the antibiotic treatment is removed. These conceptual advances raise the possibility that successful targeting of m1 G37-tRNA methylation can provide new approaches for treating acute and chronic infections caused by Gram-negative bacteria. This article is categorized under: Translation > Translation Regulation RNA Processing > RNA Editing and Modification RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

7.
Cell Syst ; 8(4): 302-314.e8, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30981730

RESUMO

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m1G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m1G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m1G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.


Assuntos
Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Metilação , RNA de Transferência/genética , Salmonella , Transcriptoma , tRNA Metiltransferases/metabolismo
8.
Sci Rep ; 8(1): 7229, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739985

RESUMO

Apples are well known to have various benefits for the human body. Procyanidins are a class of polyphenols found in apples that have demonstrated effects on the circulatory system and skeletal organs. Osteoarthritis (OA) is a locomotive syndrome that is histologically characterized by cartilage degeneration associated with the impairment of proteoglycan homeostasis in chondrocytes. However, no useful therapy for cartilage degeneration has been developed to date. In the present study, we detected beneficial effects of apple polyphenols or their procyanidins on cartilage homeostasis. An in vitro assay revealed that apple polyphenols increased the activities of mitochondrial dehydrogenases associated with an increased copy number of mitochondrial DNA as well as the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), suggesting the promotion of PGC-1α-mediated mitochondrial biogenesis. Apple  procyanidins also enhanced proteoglycan biosynthesis with aggrecan upregulation in primary chondrocytes. Of note, oral treatment with apple procyanidins prevented articular cartilage degradation in OA model mice induced by mitochondrial dysfunction in chondrocytes. Our findings suggest that apple procyanidins are promising food components that inhibit OA progression by promoting mitochondrial biogenesis and proteoglycan homeostasis in chondrocytes.


Assuntos
Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Osteoartrite/prevenção & controle , Polifenóis/farmacologia , Proantocianidinas/farmacologia , Proteoglicanas/biossíntese , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Linhagem Celular , Condrócitos/metabolismo , Condrócitos/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Masculino , Malus/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Biogênese de Organelas , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Paraquat/antagonistas & inibidores , Paraquat/toxicidade , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Polifenóis/isolamento & purificação , Cultura Primária de Células , Proantocianidinas/isolamento & purificação , Proteoglicanas/agonistas , Proteoglicanas/genética , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética
9.
Nucleic Acids Res ; 46(7): e37, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29361055

RESUMO

Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.


Assuntos
Anticódon/genética , Conformação de Ácido Nucleico , RNA de Transferência de Prolina/genética , RNA de Transferência/genética , Sequência de Bases , Códon/genética , Escherichia coli/genética , Metilação , RNA Bacteriano/genética , Uridina/análogos & derivados , Uridina/genética
10.
Front Genet ; 9: 713, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30687389

RESUMO

Although the genetic code is degenerate, synonymous codons for the same amino acid are not translated equally. Codon-specific translation is important for controlling gene expression and determining the proteome of a cell. At the molecular level, codon-specific translation is regulated by post-transcriptional epigenetic modifications of tRNA primarily at the wobble position 34 and at position 37 on the 3'-side of the anticodon. Modifications at these positions determine the quality of codon-anticodon pairing and the speed of translation on the ribosome. Different modifications operate in distinct mechanisms of codon-specific translation, generating a diversity of regulation that is previously unanticipated. Here we summarize recent work that demonstrates codon-specific translation mediated by the m1G37 methylation of tRNA at CCC and CCU codons for proline, an amino acid that has unique features in translation.

11.
ACS Pharmacol Transl Sci ; 1(1): 21-31, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-32219202

RESUMO

Although abnormal increases in the level or activity of cyclin-dependent kinase 4 (CDK4) occur frequently in cancer, the underlying mechanism is not fully understood. Here, we show that methionyl-tRNA synthetase (MRS) specifically stabilizes CDK4 by enhancing the formation of the complex between CDK4 and a chaperone protein. Knockdown of MRS reduced the CDK4 level, resulting in G0/G1 cell cycle arrest. The effects of MRS on CDK4 stability were more prominent in the tumor suppressor p16INK4a-negative cancer cells because of the competitive relationship of the two proteins for binding to CDK4. Suppression of MRS reduced cell transformation and the tumorigenic ability of a p16INK4a-negative breast cancer cell line in vivo. Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16INK4a-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.

12.
Enzymes ; 41: 89-115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28601227

RESUMO

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated m1G37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of m1G37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg2+ in the catalytic mechanism. This Mg2+ dependence is important for regulating Mg2+ transport to Salmonella for survival of the pathogen in the host cell. The strict conservation of TrmD among bacterial species suggests that a better characterization of its enzymology and biology will have a broad impact on our understanding of bacterial pathogenesis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metilação , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Especificidade por Substrato
13.
Nucleic Acids Res ; 45(7): 4081-4093, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-27956502

RESUMO

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.


Assuntos
Aptâmeros de Nucleotídeos/química , Biossíntese de Proteínas , Sondas RNA/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Compostos de Benzil/química , Escherichia coli/genética , Corantes Fluorescentes , Imidazolinas/química , Microscopia de Fluorescência , Ribossomos/metabolismo , Spinacia oleracea/genética
14.
Proc Natl Acad Sci U S A ; 113(52): 15096-15101, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-27849575

RESUMO

In Salmonella enterica serovar Typhimurium, Mg2+ limitation induces transcription of the mgtA Mg2+ transport gene, but the mechanism involved is unclear. The 5' leader of the mgtA mRNA contains a 17-codon, proline-rich ORF, mgtL, whose translation regulates the transcription of mgtA [Park S-Y et al. (2010) Cell 142:737-748]. Rapid translation of mgtL promotes formation of a secondary structure in the mgtA mRNA that permits termination of transcription by the Rho protein upstream of mgtA, whereas slow or incomplete translation of mgtL generates a different structure that blocks termination. We identified the following mutations that conferred high-level transcription of mgtA at high [Mg2+]: (i) a base-pair change that introduced an additional proline codon into mgtL, generating three consecutive proline codons; (ii) lesions in rpmA and rpmE, which encode ribosomal proteins L27 and L31, respectively; (iii) deletion of efp, which encodes elongation factor EF-P that assists the translation of proline codons; and (iv) a heat-sensitive mutation in trmD, whose product catalyzes the m1G37 methylation of tRNAPro Furthermore, substitution of three of the four proline codons in mgtL rendered mgtA uninducible. We hypothesize that the proline codons present an impediment to the translation of mgtL, which can be alleviated by high [Mg2+] exerted on component(s) of the translation machinery, such as EF-P, TrmD, or a ribosomal factor. Inadequate [Mg2+] precludes this alleviation, making mgtL translation inefficient and thereby permitting mgtA transcription. These findings are a significant step toward defining the target of Mg2+ in the regulation of mgtA transcription.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Magnésio/química , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/química , Prolina/química , Salmonella typhimurium/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Códon , Proteínas de Escherichia coli/química , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mutação , Fatores de Alongamento de Peptídeos/química , Peptídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas , RNA de Transferência/química , Ribossomos/química , Ribossomos/metabolismo , Transcrição Genética/efeitos dos fármacos , tRNA Metiltransferases/química
15.
Methods Enzymol ; 560: 91-116, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26253967

RESUMO

Transfer RNA (tRNA) molecules contain many chemical modifications that are introduced after transcription. A major form of these modifications is methyl transfer to bases and backbone groups, using S-adenosyl methionine (AdoMet) as the methyl donor. Each methylation confers a specific advantage to tRNA in structure or in function. A remarkable methylation is to the G37 base on the 3'-side of the anticodon to generate m(1)G37-tRNA, which suppresses frameshift errors during protein synthesis and is therefore essential for cell growth in all three domains of life. This methylation is catalyzed by TrmD in bacteria and by Trm5 in eukaryotes and archaea. Although TrmD and Trm5 catalyze the same methylation reaction, kinetic analysis reveals that these two enzymes are unrelated to each other and are distinct in their reaction mechanism. This chapter summarizes the kinetic assays that are used to reveal the distinction between TrmD and Trm5. Three types of assays are described, the steady-state, the pre-steady-state, and the single-turnover assays, which collectively provide the basis for mechanistic investigation of AdoMet-dependent methyl transfer reactions.


Assuntos
Proteínas de Escherichia coli/química , RNA de Transferência/química , tRNA Metiltransferases/química , Catálise , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Metilação , RNA de Transferência/metabolismo , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , tRNA Metiltransferases/metabolismo
16.
Proc Natl Acad Sci U S A ; 112(31): E4197-205, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26183229

RESUMO

The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Haemophilus influenzae/enzimologia , RNA de Transferência/metabolismo , Thermotoga maritima/enzimologia , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Sequência de Aminoácidos , Anticódon/genética , Sequência de Bases , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Guanina/metabolismo , Cinética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , RNA de Transferência/química , RNA de Transferência/genética , S-Adenosilmetionina , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
17.
Int J Mol Sci ; 16(7): 14866-83, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26140378

RESUMO

Native tRNAs often contain post-transcriptional modifications to the wobble position to expand the capacity of reading the genetic code. Some of these modifications, due to the ability to confer imperfect codon-anticodon pairing at the wobble position, can induce a high propensity for tRNA to shift into alternative reading frames. An example is the native UGG isoacceptor of E. coli tRNAPro whose wobble nucleotide U34 is post-transcriptionally modified to cmo5U34 to read all four proline codons (5'-CCA, 5'-CCC, 5'-CCG, and 5'-CCU). Because the pairing of the modified anticodon to CCC codon is particularly weak relative to CCA and CCG codons, this tRNA can readily shift into both the +1 and +2-frame on the slippery mRNA sequence CCC-CG. We show that the shift to the +2-frame is more dominant, driven by the higher stability of the codon-anticodon pairing at the wobble position. Kinetic analysis suggests that both types of shifts can occur during stalling of the tRNA in a post-translocation complex or during translocation from the A to the P-site. Importantly, while the +1-frame post complex is active for peptidyl transfer, the +2-frame complex is a poor peptidyl donor. Together with our recent work, we draw a mechanistic distinction between +1 and +2-frameshifts, showing that while the +1-shifts are suppressed by the additional post-transcriptionally modified m1G37 nucleotide in the anticodon loop, the +2-shifts are suppressed by the ribosome, supporting a role of the ribosome in the overall quality control of reading-frame maintenance.


Assuntos
Mutação da Fase de Leitura , RNA de Transferência de Prolina/genética , Pareamento de Bases , Códon/genética , Escherichia coli/genética , RNA Mensageiro/genética
18.
Nat Commun ; 6: 7226, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-26009254

RESUMO

Maintaining the translational reading frame poses difficulty for the ribosome. Slippery mRNA sequences such as CC[C/U]-[C/U], read by isoacceptors of tRNA(Pro), are highly prone to +1 frameshift (+1FS) errors. Here we show that +1FS errors occur by two mechanisms, a slow mechanism when tRNA(Pro) is stalled in the P-site next to an empty A-site and a fast mechanism during translocation of tRNA(Pro) into the P-site. Suppression of +1FS errors requires the m(1)G37 methylation of tRNA(Pro) on the 3' side of the anticodon and the translation factor EF-P. Importantly, both m(1)G37 and EF-P show the strongest suppression effect when CC[C/U]-[C/U] are placed at the second codon of a reading frame. This work demonstrates that maintaining the reading frame immediately after the initiation of translation by the ribosome is an essential aspect of protein synthesis.


Assuntos
Mutação da Fase de Leitura , Biossíntese de Proteínas , Fases de Leitura , Ribossomos/metabolismo , Escherichia coli , Fatores de Alongamento de Peptídeos/metabolismo
19.
J Biol Chem ; 289(47): 32729-41, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25288793

RESUMO

N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA(Met)) for both initiation and elongation. A portion of Met-tRNA(Met) is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.


Assuntos
Hidroximetil e Formil Transferases/genética , Doença de Leigh/genética , Proteínas Mitocondriais/genética , Mutação , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Hidroximetil e Formil Transferases/metabolismo , Immunoblotting , Doença de Leigh/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Biossíntese de Proteínas/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo
20.
J Biol Chem ; 288(40): 28987-96, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-23986443

RESUMO

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Mutação/genética , RNA de Transferência/metabolismo , Temperatura , tRNA Metiltransferases/genética , Alelos , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fluorescência , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Desnaturação Proteica , Especificidade por Substrato , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
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