Journal articles on the topic 'TRNA Function'
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1
Mangroo, Dev, Xin-Qi Wu, and Uttam L. Rajbhandary. "Escherichia coliinitiator tRNA: structure–function relationships and interactions with the translational machinery." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1023–31. http://dx.doi.org/10.1139/o95-109.
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We showed previously that the sequence and (or) structural elements important for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anticodon stem and loop. This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tRNAs generated during this work. First, we have studied the effect of overproduction of methionyl-tRNA transformylase (MTF) and initiation factors IF2 and IF3 on activity of mutant initiator tRNAs mat are defective at specific steps in the initiation pathway. Overproduction of MTF rescued specifically the activity of mutant tRNAs defective in formylation but not mutants defective in binding to the P site. Overproduction of IF2 increased me activity of all mutant tRNAs having the CUA anticodon but not of mutant tRNA having me GAC anticodon. Overproduction of IF3 had no effect on the activity of any of me mutant tRNAs tested. Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU→CUA anticodon sequence mutant mat can initiate protein synthesis from UAG instead of AUG. In contrast with me wild-type initiator tRNA, the mutant initiator tRNA has a 2-methylthio-N6-isopentenyl adenosine (ms2i6A) base modification next to the anticodon. Interestingly, this base modification is now important for activity of the mutant tRNA in initiation. In a miaA strain of E. coli deficient in biosynthesis of ms2i6A, the mutant initiator tRNA is much less active in initiation. The defect is specifically in binding to the ribosomal P site.Key words: initiator tRNA, initiation Factors, formylation, P site binding, base modification.
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Schaffer, Ashleigh E., Otis Pinkard, and Jeffery M. Coller. "tRNA Metabolism and Neurodevelopmental Disorders." Annual Review of Genomics and Human Genetics 20, no. 1 (August 31, 2019): 359–87. http://dx.doi.org/10.1146/annurev-genom-083118-015334.
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tRNAs are short noncoding RNAs required for protein translation. The human genome includes more than 600 putative tRNA genes, many of which are considered redundant. tRNA transcripts are subject to tightly controlled, multistep maturation processes that lead to the removal of flanking sequences and the addition of nontemplated nucleotides. Furthermore, tRNAs are highly structured and posttranscriptionally modified. Together, these unique features have impeded the adoption of modern genomics and transcriptomics technologies for tRNA studies. Nevertheless, it has become apparent from human neurogenetic research that many tRNA biogenesis proteins cause brain abnormalities and other neurological disorders when mutated. The cerebral cortex, cerebellum, and peripheral nervous system show defects, impairment, and degeneration upon tRNA misregulation, suggesting that they are particularly sensitive to changes in tRNA expression or function. An integrated approach to identify tRNA species and contextually characterize tRNA function will be imperative to drive future tool development and novel therapeutic design for tRNA-associated disorders.
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Francis, M. A., and U. L. Rajbhandary. "Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 9 (September 1990): 4486–94. http://dx.doi.org/10.1128/mcb.10.9.4486-4494.1990.
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We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.
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Francis, M. A., and U. L. Rajbhandary. "Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 9 (September 1990): 4486–94. http://dx.doi.org/10.1128/mcb.10.9.4486.
Full textAbstract:
We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.
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von Pawel-Rammingen, U., S. Aström, and A. S. Byström. "Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 4 (April 1992): 1432–42. http://dx.doi.org/10.1128/mcb.12.4.1432-1442.1992.
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The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.
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von Pawel-Rammingen, U., S. Aström, and A. S. Byström. "Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 4 (April 1992): 1432–42. http://dx.doi.org/10.1128/mcb.12.4.1432.
Full textAbstract:
The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.
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Strobel, M. C., and J. Abelson. "Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo." Molecular and Cellular Biology 6, no. 7 (July 1986): 2663–73. http://dx.doi.org/10.1128/mcb.6.7.2663-2673.1986.
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The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.
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Strobel, M. C., and J. Abelson. "Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo." Molecular and Cellular Biology 6, no. 7 (July 1986): 2663–73. http://dx.doi.org/10.1128/mcb.6.7.2663.
Full textAbstract:
The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.
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Oberbauer, Vera, and Matthias Schaefer. "tRNA-Derived Small RNAs: Biogenesis, Modification, Function and Potential Impact on Human Disease Development." Genes 9, no. 12 (December 5, 2018): 607. http://dx.doi.org/10.3390/genes9120607.
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Transfer RNAs (tRNAs) are abundant small non-coding RNAs that are crucially important for decoding genetic information. Besides fulfilling canonical roles as adaptor molecules during protein synthesis, tRNAs are also the source of a heterogeneous class of small RNAs, tRNA-derived small RNAs (tsRNAs). Occurrence and the relatively high abundance of tsRNAs has been noted in many high-throughput sequencing data sets, leading to largely correlative assumptions about their potential as biologically active entities. tRNAs are also the most modified RNAs in any cell type. Mutations in tRNA biogenesis factors including tRNA modification enzymes correlate with a variety of human disease syndromes. However, whether it is the lack of tRNAs or the activity of functionally relevant tsRNAs that are causative for human disease development remains to be elucidated. Here, we review the current knowledge in regard to tsRNAs biogenesis, including the impact of RNA modifications on tRNA stability and discuss the existing experimental evidence in support for the seemingly large functional spectrum being proposed for tsRNAs. We also argue that improved methodology allowing exact quantification and specific manipulation of tsRNAs will be necessary before developing these small RNAs into diagnostic biomarkers and when aiming to harness them for therapeutic purposes.
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Tucker, Jessica M., Aaron M. Schaller, Ian Willis, and Britt A. Glaunsinger. "Alteration of the Premature tRNA Landscape by Gammaherpesvirus Infection." mBio 11, no. 6 (December 15, 2020): e02664-20. http://dx.doi.org/10.1128/mbio.02664-20.
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ABSTRACTTransfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome, yet their expression and noncanonical function remain understudied. Many DNA tumor viruses enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome-wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 induces global changes in premature tRNA (pre-tRNA) expression, with 14% of tRNA genes upregulated more than 3-fold, indicating that differential tRNA gene induction is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to increased RNAPIII occupancy for the subset of tRNA genes tested; additionally, posttranscriptional mechanisms contribute to the accumulation of pre-tRNA species. We find increased abundance of tRNA fragments derived from pre-tRNAs upregulated by viral infection, suggesting that noncanonical tRNA cleavage is also affected. Furthermore, pre-tRNA accumulation, but not RNAPIII recruitment, requires gammaherpesvirus-induced degradation of host mRNAs by the virally encoded mRNA endonuclease muSOX. We hypothesize that depletion of pre-tRNA maturation or turnover machinery contributes to robust accumulation of full-length pre-tRNAs in infected cells. Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus infection and highlight the potential of using viruses to explore tRNA biology.IMPORTANCE Viral infection can dramatically change the gene expression landscape of the host cell, yet little is known regarding changes in noncoding gene transcription by RNA polymerase III (RNAPIII). Among these are transfer RNAs (tRNAs), which are fundamental in protein translation, yet whose gene regulatory features remain largely undefined in mammalian cells. Here, we perform the first genome-wide analysis of tRNA expression changes during viral infection. We show that premature tRNAs accumulate during infection with the model gammaherpesvirus MHV68 as a consequence of increased transcription, but that transcripts do not undergo canonical maturation into mature tRNAs. These findings underscore how tRNA expression is a highly regulated process, especially during conditions of elevated RNAPIII activity.
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Willis, Ian M., and Robyn D. Moir. "Signaling to and from the RNA Polymerase III Transcription and Processing Machinery." Annual Review of Biochemistry 87, no. 1 (June 20, 2018): 75–100. http://dx.doi.org/10.1146/annurev-biochem-062917-012624.
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RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.
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Calvo, Olga, Rafael Cuesta, James Anderson, Noelia Gutiérrez, Minerva Teresa García-Barrio, Alan G. Hinnebusch, and Mercedes Tamame. "GCD14p, a Repressor of GCN4 Translation, Cooperates with Gcd10p and Lhp1p in the Maturation of Initiator Methionyl-tRNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4167–81. http://dx.doi.org/10.1128/mcb.19.6.4167.
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ABSTRACT Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs forS-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAi Met) precursors containing 5′ and 3′ extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAi Met (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3′-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNAi Met. A mutation inGCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAi Met in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts.
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Arroyo, Maria Nicol, Jonathan Alex Green, Miriam Cnop, and Mariana Igoillo-Esteve. "tRNA Biology in the Pathogenesis of Diabetes: Role of Genetic and Environmental Factors." International Journal of Molecular Sciences 22, no. 2 (January 6, 2021): 496. http://dx.doi.org/10.3390/ijms22020496.
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The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes.
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Andersen, Kasper L., and Kathleen Collins. "Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena." Molecular Biology of the Cell 23, no. 1 (January 2012): 36–44. http://dx.doi.org/10.1091/mbc.e11-08-0689.
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RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A–RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.
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Huber, Sabrina, Andrea Leonardi, Peter Dedon, and Thomas Begley. "The Versatile Roles of the tRNA Epitranscriptome during Cellular Responses to Toxic Exposures and Environmental Stress." Toxics 7, no. 1 (March 25, 2019): 17. http://dx.doi.org/10.3390/toxics7010017.
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Living organisms respond to environmental changes and xenobiotic exposures by regulating gene expression. While heat shock, unfolded protein, and DNA damage stress responses are well-studied at the levels of the transcriptome and proteome, tRNA-mediated mechanisms are only recently emerging as important modulators of cellular stress responses. Regulation of the stress response by tRNA shows a high functional diversity, ranging from the control of tRNA maturation and translation initiation, to translational enhancement through modification-mediated codon-biased translation of mRNAs encoding stress response proteins, and translational repression by stress-induced tRNA fragments. tRNAs need to be heavily modified post-transcriptionally for full activity, and it is becoming increasingly clear that many aspects of tRNA metabolism and function are regulated through the dynamic introduction and removal of modifications. This review will discuss the many ways that nucleoside modifications confer high functional diversity to tRNAs, with a focus on tRNA modification-mediated regulation of the eukaryotic response to environmental stress and toxicant exposures. Additionally, the potential applications of tRNA modification biology in the development of early biomarkers of pathology will be highlighted.
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Ruan, B., I. Ahel, A. Ambrogelly, H. D. Becker, S. Bunjun, L. Feng, D. Tumbula-Hansen, et al. "Genomics and the evolution of aminoacyl-tRNA synthesis." Acta Biochimica Polonica 48, no. 2 (June 30, 2001): 313–21. http://dx.doi.org/10.18388/abp.2001_3917.
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Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.
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Strub, Benjamin R., Manoja B. K. Eswara, Jacqueline B. Pierce, and Dev Mangroo. "Utp8p Is a Nucleolar tRNA-binding Protein That Forms a Complex with Components of the Nuclear tRNA Export Machinery in Saccharomyces cerevisiae." Molecular Biology of the Cell 18, no. 10 (October 2007): 3845–59. http://dx.doi.org/10.1091/mbc.e06-11-1016.
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Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.
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ROVNER, SOPHIE. "NEW FUNCTION FOR tRNA." Chemical & Engineering News 88, no. 12 (March 22, 2010): 12. http://dx.doi.org/10.1021/cen-v088n012.p012a.
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Ding, Yu, Beibei Gao, and Jinyu Huang. "Mitochondrial Cardiomyopathy: The Roles of mt-tRNA Mutations." Journal of Clinical Medicine 11, no. 21 (October 30, 2022): 6431. http://dx.doi.org/10.3390/jcm11216431.
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Mitochondria are important organelles whose primary role is generating energy through the oxidative phosphorylation (OXPHOS) system. Cardiomyopathy, a common clinical disorder, is frequently associated with pathogenic mutations in nuclear and mitochondrial genes. To date, a growing number of nuclear gene mutations have been linked with cardiomyopathy; however, knowledge about mitochondrial tRNAs (mt-tRNAs) mutations in this disease remain inadequately understood. In fact, defects in mt-tRNA metabolism caused by pathogenic mutations may influence the functioning of the OXPHOS complexes, thereby impairing mitochondrial translation, which plays a critical role in the predisposition of this disease. In this review, we summarize some basic knowledge about tRNA biology, including its structure and function relations, modification, CCA-addition, and tRNA import into mitochondria. Furthermore, a variety of molecular mechanisms underlying tRNA mutations that cause mitochondrial dysfunctions are also discussed in this article.
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Yang, Wen-Qing, Qing-Ping Xiong, Jian-Yang Ge, Hao Li, Wen-Yu Zhu, Yan Nie, Xiuying Lin, et al. "THUMPD3–TRMT112 is a m2G methyltransferase working on a broad range of tRNA substrates." Nucleic Acids Research 49, no. 20 (October 20, 2021): 11900–11919. http://dx.doi.org/10.1093/nar/gkab927.
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Abstract Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m2G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m2G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3–TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3′-CCA of mature tRNAs. We also showed that m2G7 of tRNATrp was introduced by THUMPD3–TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3-knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m2G6/7 modification and pave a way for further studies of the role of m2G in sperm tRNA derived fragments.
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Mohanty, Bijoy K., Ankit Agrawal, and Sidney R. Kushner. "Generation of pre-tRNAs from polycistronic operons is the essential function of RNase P in Escherichia coli." Nucleic Acids Research 48, no. 5 (January 29, 2020): 2564–78. http://dx.doi.org/10.1093/nar/gkz1188.
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Abstract Ribonuclease P (RNase P) is essential for the 5′-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5′-immature tRNAs can be aminoacylated unless their 5′-extensions ≥8 nt. Surprisingly, the failure of 5′-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5′-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.
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Koltun, Bella, Sivan Ironi, Noga Gershoni-Emek, Iliana Barrera, Mohammad Hleihil, Siddharth Nanguneri, Ranjan Sasmal, Sarit S. Agasti, Deepak Nair, and Kobi Rosenblum. "Measuring mRNA translation in neuronal processes and somata by tRNA-FRET." Nucleic Acids Research 48, no. 6 (January 24, 2020): e32-e32. http://dx.doi.org/10.1093/nar/gkaa042.
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Abstract In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.
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Keeney, J. B., K. B. Chapman, V. Lauermann, D. F. Voytas, S. U. Aström, U. von Pawel-Rammingen, A. Byström, and J. D. Boeke. "Multiple molecular determinants for retrotransposition in a primer tRNA." Molecular and Cellular Biology 15, no. 1 (January 1995): 217–26. http://dx.doi.org/10.1128/mcb.15.1.217.
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Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.
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Fu, Dragony, Jennifer A. N. Brophy, Clement T. Y. Chan, Kyle A. Atmore, Ulrike Begley, Richard S. Paules, Peter C. Dedon, Thomas J. Begley, and Leona D. Samson. "Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival." Molecular and Cellular Biology 30, no. 10 (March 22, 2010): 2449–59. http://dx.doi.org/10.1128/mcb.01604-09.
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ABSTRACT tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm5U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm5U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.
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Chafe, Shawn C., and Dev Mangroo. "Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex." Molecular Biology of the Cell 21, no. 14 (July 15, 2010): 2483–99. http://dx.doi.org/10.1091/mbc.e10-03-0176.
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Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.
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Wu, Jingyan, Alicia Bao, Kunal Chatterjee, Yao Wan, and Anita K. Hopper. "Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics." Genes & Development 29, no. 24 (December 15, 2015): 2633–44. http://dx.doi.org/10.1101/gad.269803.115.
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Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology.
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Noller, Harry F., Rachel Green, Gabriele Heilek, Vernita Hoffarth, Alexander Hüttenhofer, Simpson Joseph, Inho Lee, et al. "Structure and function of ribosomal RNA." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 997–1009. http://dx.doi.org/10.1139/o95-107.
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A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.
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Wang, Jin-Tao, Jing-Bo Zhou, Xue-Ling Mao, Li Zhou, Meirong Chen, Wenhua Zhang, En-Duo Wang, and Xiao-Long Zhou. "Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs." Nucleic Acids Research 50, no. 4 (February 1, 2022): 2223–39. http://dx.doi.org/10.1093/nar/gkac056.
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Abstract N 6-Threonylcarbamoyladenosine (t6A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t6A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t6A moiety. Using t6A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNAMet and modification enzymes. The role of the universal CCA end in t6A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNAIle aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t6A functions as a tRNAIle isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t6A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t6A in tRNAIle aminoacylation and codon decoding in human cells.
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Shikha, Shikha, Rebecca Brogli, André Schneider, and Norbert Polacek. "tRNA Biology in Trypanosomes." CHIMIA International Journal for Chemistry 73, no. 5 (May 29, 2019): 395–405. http://dx.doi.org/10.2533/chimia.2019.395.
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Besides their medical importance, the parasitic protozoan Trypanosoma brucei and its relatives are experimentally highly accessible model systems for many cell biological processes. Trypanosomes are phylogenetically essentially unrelated to the popular model eukaryotes, such as yeast and animals, and thus show several unique features, many of which are connected to RNA. Here we review the tRNA biology of trypanosomes. Even though tRNAs were already discovered 60 years ago, owing to current technological advances in the field, research on tRNA biology has seen a Renaissance in recent years. First we discuss the extensive mitochondrial tRNA import process and the consequences it has for the parasite. Next we focus on trypanosomal aminoacyl-tRNA synthetases, some of which may be exploited as drug targets. Furthermore, we summarize what is known about trypanosomal tRNA modifications in both the cytosol and the mitochondrion. Finally, we provide an overview on the emerging field of tRNA-derived fragments and their possible function as translation regulators.
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Hayne, Cassandra K., Casey A. Schmidt, Maira I. Haque, A. Gregory Matera, and Robin E. Stanley. "Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage." Nucleic Acids Research 48, no. 14 (June 1, 2020): 7609–22. http://dx.doi.org/10.1093/nar/gkaa438.
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Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.
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Shikha, Shikha, and André Schneider. "The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria." Journal of Biological Chemistry 295, no. 18 (March 31, 2020): 6138–50. http://dx.doi.org/10.1074/jbc.ra119.011877.
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tRNAs universally carry a CCA nucleotide triplet at their 3′-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.
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Lee, Jin-Ok, Minho Lee, and Yeun-Jun Chung. "DBtRend: A Web-Server of tRNA Expression Profiles from Small RNA Sequencing Data in Humans." Genes 12, no. 10 (October 3, 2021): 1576. http://dx.doi.org/10.3390/genes12101576.
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Transfer RNA (tRNA), a key component of the translation machinery, plays critical roles in stress conditions and various diseases. While knowledge regarding the importance of tRNA function is increasing, its biological roles are still not well understood. There is currently no comprehensive database or web server providing the expression landscape of tRNAs across a variety of human tissues and diseases. Here, we constructed a user-friendly and interactive database, DBtRend, which provides a profile of mature tRNA expression across various biological conditions by reanalyzing the small RNA or microRNA sequencing data from the Cancer Genome Atlas (TCGA) and NCBI’s Gene Expression Omnibus (GEO) in humans. Users can explore not only the expression values of mature individual tRNAs in the human genome, but also those of isodecoders and isoacceptors based on our specific pipelines. DBtRend provides the expressed patterns of tRNAs, the differentially expressed tRNAs in different biological conditions, and the information of samples or patients, tissue types, and molecular subtype of cancers. The database is expected to help researchers interested in functional discoveries of tRNAs.
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Yermovsky-Kammerer, Audra E., and Stephen L. Hajduk. "In Vitro Import of a Nuclearly Encoded tRNA into the Mitochondrion of Trypanosoma brucei." Molecular and Cellular Biology 19, no. 9 (September 1, 1999): 6253–59. http://dx.doi.org/10.1128/mcb.19.9.6253.
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ABSTRACT All of the mitochondrial tRNAs of Trypanosoma bruceihave been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. An in vitro import assay has been developed to study the pathway of tRNA import inT. brucei. The in vitro system utilizes crude isolated trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-encoded tRNA gene cluster. The substrate, composed of tRNASer and tRNALeu, is transcribed in tandem with a 59-nucleotide intergenic region. The tandem tRNA substrate is imported rapidly, while the mature-size tRNALeu fails to be imported in this system. These results suggest that the preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits import of the tandem tRNA substrate. Characterization of the import requirements indicates that mitochondrial RNA import proceeds by a pathway including a protein component associated with the outer mitochondrial membrane, ATP-dependent steps, and a mitochondrial membrane potential.
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Gagnon, Matthieu G., Jinzhong Lin, and Thomas A. Steitz. "Elongation factor 4 remodels the A-site tRNA on the ribosome." Proceedings of the National Academy of Sciences 113, no. 18 (April 18, 2016): 4994–99. http://dx.doi.org/10.1073/pnas.1522932113.
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During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.
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Wei, Fan-Yan, and Kazuhito Tomizawa. "tRNA modifications and islet function." Diabetes, Obesity and Metabolism 20 (September 2018): 20–27. http://dx.doi.org/10.1111/dom.13405.
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Kuhn, Claus-D. "RNA versatility governs tRNA function." BioEssays 38, no. 5 (March 18, 2016): 465–73. http://dx.doi.org/10.1002/bies.201500190.
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Schultz, Dennis W., and Michael Yarus. "tRNA Structure and Ribosomal Function." Journal of Molecular Biology 235, no. 5 (February 1994): 1381–94. http://dx.doi.org/10.1006/jmbi.1994.1095.
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Schultz, Dennis W., and Michael Yarus. "tRNA Structure and Ribosomal Function." Journal of Molecular Biology 235, no. 5 (February 1994): 1395–405. http://dx.doi.org/10.1006/jmbi.1994.1096.
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Su, Chenchen, Mengqi Jin, and Wenhua Zhang. "Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life." International Journal of Molecular Sciences 23, no. 21 (November 6, 2022): 13600. http://dx.doi.org/10.3390/ijms232113600.
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The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon–codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3−/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD–TsaB –TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway–Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure–function relationship from perspectives of conservation and diversity.
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Liu, Ziwei, Oscar Vargas-Rodriguez, Yuki Goto, Eva Maria Novoa, Lluís Ribas de Pouplana, Hiroaki Suga, and Karin Musier-Forsyth. "Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation." Proceedings of the National Academy of Sciences 112, no. 19 (April 27, 2015): 6027–32. http://dx.doi.org/10.1073/pnas.1423664112.
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Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNAPro and Cys-tRNAPro, respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed “ProXp-ST1” and “ProXp-ST2,” respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.
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Dauden, Maria I., Marcin Jaciuk, Felix Weis, Ting-Yu Lin, Carolin Kleindienst, Nour El Hana Abbassi, Heena Khatter, et al. "Molecular basis of tRNA recognition by the Elongator complex." Science Advances 5, no. 7 (July 2019): eaaw2326. http://dx.doi.org/10.1126/sciadv.aaw2326.
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The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo–electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.
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JOHANSSON, MARCUS J. O., and ANDERS S. BYSTR??M. "Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation." RNA 8, no. 3 (March 2002): 324–35. http://dx.doi.org/10.1017/s1355838202027851.
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Kawabata, Mai, Kentaro Kawashima, Hiromi Mutsuro-Aoki, Tadashi Ando, Takuya Umehara, and Koji Tamura. "Peptide Bond Formation between Aminoacyl-Minihelices by a Scaffold Derived from the Peptidyl Transferase Center." Life 12, no. 4 (April 12, 2022): 573. http://dx.doi.org/10.3390/life12040573.
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The peptidyl transferase center (PTC) in the ribosome is composed of two symmetrically arranged tRNA-like units that contribute to peptide bond formation. We prepared units of the PTC components with putative tRNA-like structure and attempted to obtain peptide bond formation between aminoacyl-minihelices (primordial tRNAs, the structures composed of a coaxial stack of the acceptor stem on the T-stem of tRNA). One of the components of the PTC, P1c2UGGU (74-mer), formed a dimer and a peptide bond was formed between two aminoacyl-minihelices tethered by the dimeric P1c2UGGU. Peptide synthesis depended on both the existence of the dimeric P1c2UGGU and the sequence complementarity between the ACCA-3′ sequence of the minihelix. Thus, the tRNA-like structures derived from the PTC could have originated as a scaffold of aminoacyl-minihelices for peptide bond formation through an interaction of the CCA sequence of minihelices. Moreover, with the same origin, some would have evolved to constitute the present PTC of the ribosome, and others to function as present tRNAs.
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Phillips, Julie Baker, and David H. Ardell. "Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map." Journal of Molecular Evolution 89, no. 1-2 (February 2021): 103–16. http://dx.doi.org/10.1007/s00239-021-09995-z.
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AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.
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Persson, Britt C., Ólafur Ólafsson, Hans K. Lundgren, Lars Hederstedt, and Glenn R. Björk. "The ms2io6A37 Modification of tRNA inSalmonella typhimurium Regulates Growth on Citric Acid Cycle Intermediates." Journal of Bacteriology 180, no. 12 (June 15, 1998): 3144–51. http://dx.doi.org/10.1128/jb.180.12.3144-3151.1998.
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ABSTRACT The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA I,V Ser inEscherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E. coli. Mutants (miaE) of S. typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, themiaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.
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Florentz, Catherine. "Molecular Investigations on tRNAs Involved in Human Mitochondrial Disorders." Bioscience Reports 22, no. 1 (February 1, 2002): 81–98. http://dx.doi.org/10.1023/a:1016065107165.
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Over the last decade, human neurodegenerative disorders which correlate with point mutations in mitochondrial tRNA genes became more and more numerous. Both the number of mutations (more than 70) and the variety of phenotypes (cardiopathies, myopathies, encephalopathies as well as diabetes, deafness or others) render the understanding of the genotype/phenotype relationships very complex. Here we first summarize the efforts undertaken to decipher the initial impact of various mutations on the structure/function relationships of tRNAs. This includes several lines of research, namely (i) investigation of human mitochrondrial tRNA structures, (ii) comparison of disease-related and polymorphic mutations at a theoretical level, and (iii) experimental investigations of affected tRNAs in the frame of mitochondrial protein synthesis. A new approach aimed at searching for long-range effects of mitochondrial tRNA mutations on a broader global mitochondrial level will also be presented. Initial results obtained by comparative mitochondrial proteomics turn out to be very promising for deciphering unexpected molecular partners involved in the pathological status of the mitochondria.
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Dhungel, N., and A. K. Hopper. "Beyond tRNA cleavage: novel essential function for yeast tRNA splicing endonuclease unrelated to tRNA processing." Genes & Development 26, no. 5 (March 1, 2012): 503–14. http://dx.doi.org/10.1101/gad.183004.111.
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Djumagulov, Muminjon, Natalia Demeshkina, Lasse Jenner, Alexey Rozov, Marat Yusupov, and Gulnara Yusupova. "Accuracy mechanism of eukaryotic ribosome translocation." Nature 600, no. 7889 (December 1, 2021): 543–46. http://dx.doi.org/10.1038/s41586-021-04131-9.
Full textAbstract:
AbstractTranslation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon–anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon–anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a ‘pawl’ defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation.
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McFeeters, Hana, Morgan J. Gilbert, Rachel M. Thompson, William N. Setzer, Luis R. Cruz-Vera, and Robert L. McFeeters. "Inhibition of Essential Bacterial Peptidyl-tRNA Hydrolase Activity by Tropical Plant Extracts." Natural Product Communications 7, no. 8 (August 2012): 1934578X1200700. http://dx.doi.org/10.1177/1934578x1200700836.
Full textAbstract:
Peptidyl-tRNA Hydrolase (Pth) is a highly conserved, essential enzyme in bacteria. It removes the peptide portion from peptidyl-tRNA, returning free tRNAs to participate in translation. Build-up of peptidyl-tRNAs is toxic and defects in Pth function result in cell death. Herein we use in vitro activity of recombinant E. coli Pth to screen tropical plant extracts for inhibition. Multiple extracts were found to have inhibitory activity with some exhibiting different inhibitory effects depending on extraction conditions. IC50 values ranged from 0.02 to > 53.8 μg of extract per 1 unit of Pth, holding promise for in vivo screening. The inhibitory components in these extracts may serve as lead compounds for development of novel antibacterials.
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Strobel, M. C., and J. Abelson. "Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA." Molecular and Cellular Biology 6, no. 7 (July 1986): 2674–83. http://dx.doi.org/10.1128/mcb.6.7.2674-2683.1986.
Full textAbstract:
The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.