Journal articles: 'Initiator tRNAs'

1

Drabkin, Harold J., and Uttam L. RajBhandary. "Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5140–47. http://dx.doi.org/10.1128/mcb.18.9.5140.

Full text

Abstract:

ABSTRACT Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.

APA, Harvard, Vancouver, ISO, and other styles

2

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

3

Farruggio, D., J. Chaudhuri, U. Maitra, and U. L. RajBhandary. "The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2." Molecular and Cellular Biology 16, no. 8 (August 1996): 4248–56. http://dx.doi.org/10.1128/mcb.16.8.4248.

Full text

Abstract:

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

APA, Harvard, Vancouver, ISO, and other styles

4

Drabkin, Harold J., Melanie Estrella, and Uttam L. Rajbhandary. "Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1459–66. http://dx.doi.org/10.1128/mcb.18.3.1459.

Full text

Abstract:

ABSTRACT Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

APA, Harvard, Vancouver, ISO, and other styles

5

Das, Gautam, T. K. Dineshkumar, Swapna Thanedar, and Umesh Varshney. "Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli." Microbiology 151, no. 6 (June 1, 2005): 1741–50. http://dx.doi.org/10.1099/mic.0.27915-0.

Full text

Abstract:

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA1 fMet, and a distantly located metY gene encodes a variant, tRNA2 fMet. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA2 fMet with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA + host background. The mutations were observed when the plasmid-borne gene encoded tRNA2 fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA1 fMet genes in E. coli in safeguarding their intactness, are discussed.

APA, Harvard, Vancouver, ISO, and other styles

6

Starck, Shelley, Vivian Jiang, Mariana Pavon-Eternod, Sharanya Prasad, Tao Pan, and Nilabh Shastri. "Cryptic tRNAi shapes the pMHC I repertoire (100.5)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 100.5. http://dx.doi.org/10.4049/jimmunol.186.supp.100.5.

Full text

Abstract:

Abstract MHC class I molecules present peptides on the cell surface for immune surveillance of viruses and cancer. Interestingly, the peptides are encoded not only in conventional AUG-initiated translational reading frames but also in non-AUG initiated cryptic reading frames. Whether the same or distinct translational machinery is used to produce cryptic peptides at non-AUG start codons, such as CUG, is not known. Here, we show that translational initiation of antigenic precursors at cryptic CUG codons is differentially regulated by ribosomal initiation complexes that contain novel initiator tRNAs. This tRNA is distinct from Met-initiator tRNA and enhances initiation at CUG start codons. Thus, a novel tRNA -based mechanism can supply peptides for presentation by MHC I.

APA, Harvard, Vancouver, ISO, and other styles

7

Samhita, L., S. Shetty, and U. Varshney. "Unconventional initiator tRNAs sustain Escherichia coli." Proceedings of the National Academy of Sciences 109, no. 32 (July 24, 2012): 13058–63. http://dx.doi.org/10.1073/pnas.1207868109.

Full text

APA, Harvard, Vancouver, ISO, and other styles

8

Arhin, George K., Shuiyuan Shen, Henriette Irmer, Elisabetta Ullu, and Christian Tschudi. "Role of a 300-Kilodalton Nuclear Complex in the Maturation of Trypanosoma brucei Initiator Methionyl-tRNA." Eukaryotic Cell 3, no. 4 (August 2004): 893–99. http://dx.doi.org/10.1128/ec.3.4.893-899.2004.

Full text

Abstract:

ABSTRACT tRNAs are transcribed as precursors containing 5′ leader and 3′ extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNAMet in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5′ leader of initiator tRNAMet precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNAMet containing 5′ extensions but processed 3′ ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNAMet molecules with 5′ and 3′ extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNAMet. Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNAMet, 3′ trimming precedes 5′ processing.

APA, Harvard, Vancouver, ISO, and other styles

9

Crausaz Esseiva, Anne, Laurence Maréchal-Drouard, Anne Cosset, and André Schneider. "The T-Stem Determines the Cytosolic or Mitochondrial Localization of Trypanosomal tRNAsMet." Molecular Biology of the Cell 15, no. 6 (June 2004): 2750–57. http://dx.doi.org/10.1091/mbc.e03-11-0821.

Full text

Abstract:

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNAMet, all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNAMet is closely related to the imported elongator tRNAMet. Thus, the distinct localization of the two tRNAsMet must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAsMet. Furthermore, it was shown that the tRNAMet T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNAMet indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAsMet into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNAMet.

APA, Harvard, Vancouver, ISO, and other styles

10

Wu, X. Q., P. Iyengar, and U. L. RajBhandary. "Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes." EMBO Journal 15, no. 17 (September 1996): 4734–39. http://dx.doi.org/10.1002/j.1460-2075.1996.tb00850.x.

Full text

APA, Harvard, Vancouver, ISO, and other styles

11

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

12

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 text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

13

Shetty, Sunil, Souvik Bhattacharyya, and Umesh Varshney. "Is the cellular initiation of translation an exclusive property of the initiator tRNAs?" RNA Biology 12, no. 7 (May 21, 2015): 675–80. http://dx.doi.org/10.1080/15476286.2015.1043507.

Full text

APA, Harvard, Vancouver, ISO, and other styles

14

O'CONNOR, MICHAEL, STEVEN T. GREGORY, UTTAM L. RAJBHANDARY, and ALBERT E. DAHLBERG. "Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3." RNA 7, no. 7 (July 2001): 969–78. http://dx.doi.org/10.1017/s1355838201010184.

Full text

APA, Harvard, Vancouver, ISO, and other styles

15

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

16

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 text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

17

Charrière, Fabien, Timothy H. P. Tan, and André Schneider. "Mitochondrial Initiation Factor 2 ofTrypanosoma bruceiBinds Imported Formylated Elongator-type tRNAMet." Journal of Biological Chemistry 280, no. 16 (February 23, 2005): 15659–65. http://dx.doi.org/10.1074/jbc.m411581200.

Full text

Abstract:

The mitochondrion ofTrypanosoma bruceilacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNAMet, all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNAMetpresent inT. bruceimitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNAMetcarrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation,T. bruceihas an unusual methionyl-tRNA formyltransferase that formylates elongator tRNAMet. In the present study, we have identified initiation factor 2 ofT. bruceiand shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNAMet. Furthermore, the protein also recognizes the structurally very differentEscherichia coliinitiator tRNAMet, suggesting that the main determinant recognized is the formylated methionine.In vivostudies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclicT. brucei.

APA, Harvard, Vancouver, ISO, and other styles

18

Cevallos, Randal C., and Peter Sarnow. "Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp." Journal of Virology 79, no. 2 (January 15, 2005): 677–83. http://dx.doi.org/10.1128/jvi.79.2.677-683.2005.

Full text

Abstract:

ABSTRACT The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

APA, Harvard, Vancouver, ISO, and other styles

19

Ayyub, Shreya Ahana, Divya Dobriyal, Riyaz Ahmad Shah, Kuldeep Lahry, Madhumita Bhattacharyya, Souvik Bhattacharyya, Saikat Chakrabarti, and Umesh Varshney. "Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas." RNA Biology 15, no. 1 (September 29, 2017): 70–80. http://dx.doi.org/10.1080/15476286.2017.1377879.

Full text

APA, Harvard, Vancouver, ISO, and other styles

20

Marechal, Laurence, Pierre Guillemaut, Jean-Michel Grienenberger, Genevi�ve Jeannin, and Jacques-Henry Weil. "Sequences of initiator and elongator methionine tRNAs in bean mitochondria." Plant Molecular Biology 7, no. 4 (1986): 245–53. http://dx.doi.org/10.1007/bf00752898.

Full text

APA, Harvard, Vancouver, ISO, and other styles

21

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

22

Qiu, Hongfang, Cuihua Hu, James Anderson, Glenn R. Björk, Srimonti Sarkar, Anita K. Hopper, and Alan G. Hinnebusch. "Defects in tRNA Processing and Nuclear Export InduceGCN4 Translation Independently of Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2." Molecular and Cellular Biology 20, no. 7 (April 1, 2000): 2505–16. http://dx.doi.org/10.1128/mcb.20.7.2505-2516.2000.

Full text

Abstract:

ABSTRACT Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNAMetbinding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNAAAC Val (tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded byPUS4 also leads to translational derepression ofGCN4 (Gcd− phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd− phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate thatPUS4 overexpression did not diminish functional initiator tRNAMet levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd− phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyr that cannot be processed by RNase P had a Gcd− phenotype. Interestingly, the Gcd− phenotype of hcPUS4also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd− phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal* showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMet binding to the ribosome.

APA, Harvard, Vancouver, ISO, and other styles

23

Drabkin, H. J., B. Helk, and U. L. RajBhandary. "The role of nucleotides conserved in eukaryotic initiator methionine tRNAs in initiation of protein synthesis." Journal of Biological Chemistry 268, no. 33 (November 1993): 25221–28. http://dx.doi.org/10.1016/s0021-9258(19)74591-8.

Full text

APA, Harvard, Vancouver, ISO, and other styles

24

Li, Yan, William B. Holmes, Dean R. Appling, and Uttam L. RajBhandary. "Initiation of Protein Synthesis in Saccharomyces cerevisiae Mitochondria without Formylation of the Initiator tRNA." Journal of Bacteriology 182, no. 10 (May 15, 2000): 2886–92. http://dx.doi.org/10.1128/jb.182.10.2886-2892.2000.

Full text

Abstract:

ABSTRACT Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNAfMet) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNAfMet). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and usesN 10-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C1-tetrahydrofolate (C1-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNAfMet, Met-tRNAfMet, and tRNAfMet shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective “wild-type” parental strains. In spite of the absence of fMet-tRNAfMet, the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation ofpetite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for thefmt1 deletion strain.

APA, Harvard, Vancouver, ISO, and other styles

25

Shetty, Sunil, and Umesh Varshney. "An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation." Proceedings of the National Academy of Sciences 113, no. 41 (October 3, 2016): E6126—E6134. http://dx.doi.org/10.1073/pnas.1609550113.

Full text

Abstract:

Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.

APA, Harvard, Vancouver, ISO, and other styles

26

Tan, Timothy H. P., Roland Pach, Anne Crausaz, Al Ivens, and André Schneider. "tRNAs in Trypanosoma brucei: Genomic Organization, Expression, and Mitochondrial Import." Molecular and Cellular Biology 22, no. 11 (June 1, 2002): 3707–17. http://dx.doi.org/10.1128/mcb.22.11.3707-3716.2002.

Full text

Abstract:

ABSTRACT The mitochondrial genome of Trypanosoma brucei does not encode tRNAs. Consequently, all mitochondrial tRNAs are imported from the cytosol and originate from nucleus-encoded genes. Analysis of all currently available T. brucei sequences revealed that its genome carries 50 tRNA genes representing 40 different isoacceptors. The identified set is expected to be nearly complete since all but four codons are accounted for. The number of tRNA genes in T. brucei is very low for a eukaryote and lower than those of many prokaryotes. Using quantitative Northern analysis we have determined the absolute abundance in the cell and the mitochondrion of a group of 15 tRNAs specific for 12 amino acids. Except for the initiator type tRNAMet, which is cytosol specific, the cytosolic and the mitochondrial sets of tRNAs were qualitatively identical. However, the extent of mitochondrial localization was variable for the different tRNAs, ranging from 1 to 7.5% per cell. Finally, by using transgenic cell lines in combination with quantitative Northern analysis it was shown that import of tRNALeu(CAA) is independent of its 5′-genomic context, suggesting that the in vivo import substrate corresponds to the mature, fully processed tRNA.

APA, Harvard, Vancouver, ISO, and other styles

27

Lee, Changkeun, Gisela Kramer, David E. Graham, and Dean R. Appling. "Yeast Mitochondrial Initiator tRNA Is Methylated at Guanosine 37 by the Trm5-encoded tRNA (Guanine-N1-)-methyltransferase." Journal of Biological Chemistry 282, no. 38 (July 25, 2007): 27744–53. http://dx.doi.org/10.1074/jbc.m704572200.

Full text

Abstract:

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m1G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m1G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Δtrm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNAMetf). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNAMetf and tRNAPhe. High pressure liquid chromatography analysis showed the methylated product to be m1G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1–33 (Δ1–33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Δ1–33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m1G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.

APA, Harvard, Vancouver, ISO, and other styles

28

Vilardo, Elisa, Fabian Amman, Ursula Toth, Annika Kotter, Mark Helm, and Walter Rossmanith. "Functional characterization of the human tRNA methyltransferases TRMT10A and TRMT10B." Nucleic Acids Research 48, no. 11 (May 11, 2020): 6157–69. http://dx.doi.org/10.1093/nar/gkaa353.

Full text

Abstract:

Abstract The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.

APA, Harvard, Vancouver, ISO, and other styles

29

Tasak, Monika, and Eric M. Phizicky. "Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay path way in evolutionarily distant yeast species." PLOS Genetics 18, no. 7 (July 28, 2022): e1010215. http://dx.doi.org/10.1371/journal.pgen.1010215.

Full text

Abstract:

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5’-3’ exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3’-5’ exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.

APA, Harvard, Vancouver, ISO, and other styles

30

Bhattacharyya, Souvik, and Umesh Varshney. "Evolution of initiator tRNAs and selection of methionine as the initiating amino acid." RNA Biology 13, no. 9 (July 8, 2016): 810–19. http://dx.doi.org/10.1080/15476286.2016.1195943.

Full text

APA, Harvard, Vancouver, ISO, and other styles

31

Lemieux, Jason, Bernard Lakowski, Ashley Webb, Yan Meng, Antonio Ubach, Frédéric Bussière, Thomas Barnes, and Siegfried Hekimi. "Regulation of Physiological Rates in Caenorhabditis elegans by a tRNA-Modifying Enzyme in the Mitochondria." Genetics 159, no. 1 (September 1, 2001): 147–57. http://dx.doi.org/10.1093/genetics/159.1.147.

Full text

Abstract:

Abstract We show that the phenotype associated with gro-1(e2400) comprises the whole suite of features that characterize the phenotype of the clk mutants in Caenorhabditis elegans, including deregulated developmental, behavioral, and reproductive rates, as well as increased life span and a maternal effect. We cloned gro-1 and found that it encodes a highly conserved cellular enzyme, isopentenylpyrophosphate:tRNA transferase (IPT), which modifies a subset of tRNAs. In yeast, two forms of the enzyme are produced by alternative translation initiation, one of which is mitochondrial. In the gro-1 transcript there are also two possible initiator ATGs, between which there is a sequence predicted to encode a mitochondrial localization signal. A functional GRO-1::GFP fusion protein is localized diffusely throughout the cytoplasm and nucleus. A GRO-1::GFP initiated from the first methionine is localized exclusively to the mitochondria and rescues the mutant phenotype. In contrast, a protein initiated from the second methionine is localized diffusely throughout the cell and does not rescue the mutant phenotype. As oxygen consumption and ATP concentration have been reported to be unaffected in gro-1 mutants, our observations suggest that GRO-1 acts in mitochondria and regulates global physiology by unknown mechanisms.

APA, Harvard, Vancouver, ISO, and other styles

32

Kleiman, Lawrence, Erich Schmedt, and Harvey Miller. "The independent regulation of and tRNAAsn synthesis during Friend cell erythroid differentiation." Biochemistry and Cell Biology 66, no. 7 (July 1, 1988): 772–79. http://dx.doi.org/10.1139/o88-088.

Full text

Abstract:

In this report, we have compared the changes in the production of [Formula: see text] (initiator tRNAMet) and tRNAAsn, which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of [Formula: see text] changes very little in the cytoplasmic tRNA population, the relative concentration of tRNAAsn decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of [Formula: see text] in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNAAsn again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of [Formula: see text] (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNAAsn undergoes periodic increases and decreases in value.

APA, Harvard, Vancouver, ISO, and other styles

33

Tang, Jun, Pengfei Jia, Peiyong Xin, Jinfang Chu, Dong-Qiao Shi, and Wei-Cai Yang. "The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase." Journal of Experimental Botany 71, no. 10 (February 25, 2020): 3024–36. http://dx.doi.org/10.1093/jxb/eraa100.

Full text

Abstract:

Abstract tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.

APA, Harvard, Vancouver, ISO, and other styles

34

Drabkin, H. J., and U. L. RajBhandary. "Site-specific mutagenesis on a human initiator methionine tRNA gene within a sequence conserved in all eukaryotic initiator tRNAs and studies of its effects on in vitro transcription." Journal of Biological Chemistry 260, no. 9 (May 1985): 5580–87. http://dx.doi.org/10.1016/s0021-9258(18)89062-7.

Full text

APA, Harvard, Vancouver, ISO, and other styles

35

Seong, B. L., and U. L. RajBhandary. "Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop." Proceedings of the National Academy of Sciences 84, no. 2 (January 1, 1987): 334–38. http://dx.doi.org/10.1073/pnas.84.2.334.

Full text

APA, Harvard, Vancouver, ISO, and other styles

36

Thanedar, Swapna, N. Vinay Kumar, and Umesh Varshney. "The Fate of the Initiator tRNAs Is Sensitive to the Critical Balance between Interacting Proteins." Journal of Biological Chemistry 275, no. 27 (March 29, 2000): 20361–67. http://dx.doi.org/10.1074/jbc.m001238200.

Full text

APA, Harvard, Vancouver, ISO, and other styles

37

GROSJEAN, Henri, Suzy HENAU, Takefumi DOI, Akio YAMANE, Eiko OHTSUKA, Morio IKEHARA, Nicole BEAUCHEMIN, Krikor NICOGHOSIAN, and Robert CEDERGREN. "The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs." European Journal of Biochemistry 166, no. 2 (July 1987): 325–32. http://dx.doi.org/10.1111/j.1432-1033.1987.tb13518.x.

Full text

APA, Harvard, Vancouver, ISO, and other styles

38

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

39

Kamaike, K., H. Takahashi, K. Morohoshi, N. Kataoka, T. Kakinuma, and Y. Ishido. "Oligonucleotide synthesis using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-phosphoramidite derivatives." Acta Biochimica Polonica 45, no. 4 (December 31, 1998): 949–76. http://dx.doi.org/10.18388/abp.1998_4354.

Full text

Abstract:

A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met).

APA, Harvard, Vancouver, ISO, and other styles

40

Varshney, U., C. P. Lee, and U. L. RajBhandary. "Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase." Journal of Biological Chemistry 266, no. 36 (December 1991): 24712–18. http://dx.doi.org/10.1016/s0021-9258(18)54288-5.

Full text

APA, Harvard, Vancouver, ISO, and other styles

41

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

42

Schweisguth, David C., and Peter B. Moore. "On the conformation of the anticodon loops of initiator and elongator methionine tRNAs 1 1Edited by I. Tinoco." Journal of Molecular Biology 267, no. 3 (April 1997): 505–19. http://dx.doi.org/10.1006/jmbi.1996.0903.

Full text

APA, Harvard, Vancouver, ISO, and other styles

43

Thanedar, Swapna, T. K. Dineshkumar, and Umesh Varshney. "The Mere Lack of rT Modification in Initiator tRNA Does Not Facilitate Formylation-Independent Initiation inEscherichia coli." Journal of Bacteriology 183, no. 24 (December 15, 2001): 7397–402. http://dx.doi.org/10.1128/jb.183.24.7397-7402.2001.

Full text

Abstract:

ABSTRACT Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using trmA strains of Escherichia coli(defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.

APA, Harvard, Vancouver, ISO, and other styles

44

CIESIOLKA, Jerzy, Jan WRZESINSKI, Piotr GORNICKI, Jan PODKOWINSKI, and Wlodzimierz J. KRZYZOSIAK. "Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages." European Journal of Biochemistry 186, no. 1-2 (December 1989): 71–77. http://dx.doi.org/10.1111/j.1432-1033.1989.tb15179.x.

Full text

APA, Harvard, Vancouver, ISO, and other styles

45

Starck, Shelley R., Vivian Jiang, Mariana Pavon-Eternod, Sharanya Prasad, Brian McCarthy, Tao Pan, and Nilabh Shastri. "Leucine-tRNA Initiates at CUG Start Codons for Protein Synthesis and Presentation by MHC Class I." Science 336, no. 6089 (June 28, 2012): 1719–23. http://dx.doi.org/10.1126/science.1220270.

Full text

Abstract:

Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic non–AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNAiMet) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism allows non–AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.

APA, Harvard, Vancouver, ISO, and other styles

46

Zhang, Zhijun, Qin Yu, Sang-Moo Kang, James Buescher, and Casey D. Morrow. "Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA3Lys rather than tRNA1,2Lys." Journal of Virology 72, no. 7 (July 1, 1998): 5464–71. http://dx.doi.org/10.1128/jvi.72.7.5464-5471.1998.

Full text

Abstract:

ABSTRACT All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3 Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1 Lys, tRNA2 Lys, and tRNA3 Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3 Lys, tRNA1 Lys and tRNA2 Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3 Lys, not tRNA1 Lys or tRNA2 Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2 Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3 Lys from proviral DNA isolated fromgpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2 Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2 Lys. The results of these studies suggest that the selection of tRNA3 Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.

APA, Harvard, Vancouver, ISO, and other styles

47

Ramesh, Vaidyanathan, Caroline Köhrer, and Uttam L. RajBhandary. "Expression of Escherichia coli Methionyl-tRNA Formyltransferase in Saccharomyces cerevisiae Leads to Formylation of the Cytoplasmic Initiator tRNA and Possibly to Initiation of Protein Synthesis with Formylmethionine." Molecular and Cellular Biology 22, no. 15 (August 1, 2002): 5434–42. http://dx.doi.org/10.1128/mcb.22.15.5434-5442.2002.

Full text

Abstract:

ABSTRACT Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.

APA, Harvard, Vancouver, ISO, and other styles

48

Hartz, D., J. Binkley, T. Hollingsworth, and L. Gold. "Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3." Genes & Development 4, no. 10 (October 1, 1990): 1790–800. http://dx.doi.org/10.1101/gad.4.10.1790.

Full text

APA, Harvard, Vancouver, ISO, and other styles

49

RajBhandary, U. L. "More surprises in translation: Initiation without the initiator tRNA." Proceedings of the National Academy of Sciences 97, no. 4 (February 11, 2000): 1325–27. http://dx.doi.org/10.1073/pnas.040579197.

Full text

APA, Harvard, Vancouver, ISO, and other styles

50

Goto, Yuki, and Hiroaki Suga. "Translation Initiation with Initiator tRNA Charged with Exotic Peptides." Journal of the American Chemical Society 131, no. 14 (April 15, 2009): 5040–41. http://dx.doi.org/10.1021/ja900597d.

Full text

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Initiator tRNAs'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles