Publications by authors named "Isao Masuda"

22 Publications

  • Page 1 of 1

Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis.

Nucleic Acids Res 2021 Sep;49(17):10046-10060

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.
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http://dx.doi.org/10.1093/nar/gkab734DOI Listing
September 2021

Loss of -methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression.

Elife 2021 08 12;10. Epub 2021 Aug 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, United States.

-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of mG37 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 mG37 deficiency on protein synthesis. Using as a model, we show that mG37 deficiency induces ribosome stalling at codons that are normally translated by mG37-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 mG37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in mG37 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 mG37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
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http://dx.doi.org/10.7554/eLife.70619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384417PMC
August 2021

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation.

Nat Commun 2021 07 30;12(1):4644. Epub 2021 Jul 30.

RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA, USA.

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.
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http://dx.doi.org/10.1038/s41467-021-24911-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324841PMC
July 2021

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA.

Nat Commun 2021 01 12;12(1):328. Epub 2021 Jan 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-20373-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803779PMC
January 2021

tRNA methylation: An unexpected link to bacterial resistance and persistence to antibiotics and beyond.

Wiley Interdiscip Rev RNA 2020 11 13;11(6):e1609. Epub 2020 Jun 13.

Department of Biochemistry & Molecular Biology, and Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.

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 m G37 methylation of tRNA, at the N 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 m 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 m 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.
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http://dx.doi.org/10.1002/wrna.1609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768609PMC
November 2020

tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance.

Cell Syst 2019 04 10;8(4):302-314.e8. Epub 2019 Apr 10.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Electronic address:

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 mG37-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 mG37 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 mG37 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.
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http://dx.doi.org/10.1016/j.cels.2019.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483872PMC
April 2019

Codon-Specific Translation by mG37 Methylation of tRNA.

Front Genet 2018 10;9:713. Epub 2019 Jan 10.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, United States.

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 mG37 methylation of tRNA at CCC and CCU codons for proline, an amino acid that has unique features in translation.
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http://dx.doi.org/10.3389/fgene.2018.00713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335274PMC
January 2019

Apple procyanidins promote mitochondrial biogenesis and proteoglycan biosynthesis in chondrocytes.

Sci Rep 2018 05 8;8(1):7229. Epub 2018 May 8.

Department of Advanced Aging Medicine, Chiba University Graduate School of Medicine, Chiba, Japan.

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.
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http://dx.doi.org/10.1038/s41598-018-25348-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940809PMC
May 2018

Stabilization of Cyclin-Dependent Kinase 4 by Methionyl-tRNA Synthetase in p16-Negative Cancer.

ACS Pharmacol Transl Sci 2018 Sep 24;1(1):21-31. Epub 2018 Apr 24.

Medicinal Bioconvergence Research Center, Seoul National University, Suwon, 16229, Korea.

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 p16-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 p16-negative breast cancer cell line . Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.
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http://dx.doi.org/10.1021/acsptsci.8b00001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089025PMC
September 2018

Selective terminal methylation of a tRNA wobble base.

Nucleic Acids Res 2018 04;46(7):e37

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.
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http://dx.doi.org/10.1093/nar/gky013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909439PMC
April 2018

TrmD: A Methyl Transferase for tRNA Methylation With mG37.

Enzymes 2017;41:89-115. Epub 2017 Apr 12.

Center of New Technologies, University of Warsaw, Warsaw, Poland.

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated mG37 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 mG37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg in the catalytic mechanism. This Mg dependence is important for regulating Mg 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.
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http://dx.doi.org/10.1016/bs.enz.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054489PMC
June 2019

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

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.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

Mg2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide.

Proc Natl Acad Sci U S A 2016 12 14;113(52):15096-15101. Epub 2016 Nov 14.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907;

In Salmonella enterica serovar Typhimurium, Mg limitation induces transcription of the mgtA Mg 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 [Mg]: (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 mG37 methylation of tRNA 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 [Mg] exerted on component(s) of the translation machinery, such as EF-P, TrmD, or a ribosomal factor. Inadequate [Mg] precludes this alleviation, making mgtL translation inefficient and thereby permitting mgtA transcription. These findings are a significant step toward defining the target of Mg in the regulation of mgtA transcription.
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http://dx.doi.org/10.1073/pnas.1612268113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206563PMC
December 2016

Kinetic Analysis of tRNA Methyltransferases.

Methods Enzymol 2015 2;560:91-116. Epub 2015 Jun 2.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania USA.

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.
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http://dx.doi.org/10.1016/bs.mie.2015.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860815PMC
May 2016

Structural basis for methyl-donor-dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD.

Proc Natl Acad Sci U S A 2015 Aug 16;112(31):E4197-205. Epub 2015 Jul 16.

RIKEN Systems and Structural Biology Center, Tsurumi-ku, Yokohama 230-0045, Japan; Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Structural Biology Laboratory, Tsurumi-ku, Yokohama 230-0045, Japan

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.
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http://dx.doi.org/10.1073/pnas.1422981112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534213PMC
August 2015

The UGG Isoacceptor of tRNAPro Is Naturally Prone to Frameshifts.

Int J Mol Sci 2015 Jul 1;16(7):14866-83. Epub 2015 Jul 1.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.
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http://dx.doi.org/10.3390/ijms160714866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519876PMC
July 2015

Maintenance of protein synthesis reading frame by EF-P and m(1)G37-tRNA.

Nat Commun 2015 May 26;6:7226. Epub 2015 May 26.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.

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.
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http://dx.doi.org/10.1038/ncomms8226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445466PMC
May 2015

Biochemical characterization of pathogenic mutations in human mitochondrial methionyl-tRNA formyltransferase.

J Biol Chem 2014 Nov 6;289(47):32729-41. Epub 2014 Oct 6.

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,

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.
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http://dx.doi.org/10.1074/jbc.M114.610626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239624PMC
November 2014

The temperature sensitivity of a mutation in the essential tRNA modification enzyme tRNA methyltransferase D (TrmD).

J Biol Chem 2013 Oct 28;288(40):28987-96. Epub 2013 Aug 28.

From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

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.
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http://dx.doi.org/10.1074/jbc.M113.485797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789996PMC
October 2013

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata).

Nucleic Acids Res 2010 Oct 27;38(18):6186-94. Epub 2010 May 27.

Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.
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http://dx.doi.org/10.1093/nar/gkq449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952869PMC
October 2010

Mitochondrial group II introns in the raphidophycean flagellate Chattonella spp. suggest a diatom-to-Chattonella lateral group II intron transfer.

Protist 2009 Aug 5;160(3):364-75. Epub 2009 Apr 5.

Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.

In the cytochrome c oxidase subunit I (cox1) gene of four raphidophycean flagellates Chattonella antiqua, C. marina, C. ovata, and C. minima we found two group II introns described here as Chattonella cox1-i1 and Chattonella cox1-i2 encoding an open reading frame (ORF) comprised of three domains: reverse transcriptase (RT), RNA maturase (Ma) and zinc finger (H-N-H) endonuclease domains. The secondary structures show both Chattonella cox1-i1 and Chattonella cox1-i2 belong to group IIA1, albeit the former possesses a group IIB-like secondary structural character in the epsilon' region of arm I. Our phylogenetic analysis inferred from RT domain sequences of the intronic ORF, comparison of the insertion sites, and the secondary structures of the introns suggests that Chattonella cox1-i1 likely shares an evolutionary origin with the group II introns inserted in cox1 genes of five phylogenetically diverged eukaryotes. In contrast, Chattonella cox1-i2 was suggested to bear a close evolutionary affinity to the group II introns found in diatom cox1 genes. The RT domain-based phylogeny shows a tree topology in which Chattonella cox1-i2 is nested in the diatom sequences suggesting that a diatom-to-Chattonella intron transfer has taken place. Finally, we found no intron in cox1 genes from deeper-branching raphidophyceans. Based on parsimonious discussion, Chattonella cox1-i1 and Chattonella cox1-i2 have invaded into the cox1 gene of an ancestral Chattonella cell after diverging from C. subsalsa.
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http://dx.doi.org/10.1016/j.protis.2009.02.003DOI Listing
August 2009
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