Nucleotide sequence of rabbit liver and sheep mammary gland cytoplasmic initiator transfer RNAs

Şi̇mşek, Mehmet; RajBhandary, Uttam L.; Boisnard, Monique; Pétrissant, Guy

doi:10.1038/247518a0

Cited by 88 publications

(23 citation statements)

References 16 publications

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“…In contrast to fungi and plants (20,48,57), vertebrate initiator tRNAs do not have a 2Ј-O-phosphoribosyl modification of the ribose at position 64 (21,22,46,58,60). This raises the question of how the vertebrate initiator tRNAs are prevented from acting as elongators in the corresponding protein synthesis systems.…”

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confidence: 87%

“…This is most likely due to protrusion of the bulky group into the minor groove of the T⌿C stem helix, leading to a steric block in binding of these tRNAs to eEF1 (4,19). Further support comes from isolation of mutants of the yeast Saccharomyces cerevisiae that are defective in modification of the ribose 64 in the initiator tRNA and from the demonstration that in this mutant strain, the initiator methionine tRNA can act as an elongator in vivo (1).In contrast to fungi and plants (20,48,57), vertebrate initiator tRNAs do not have a 2Ј-O-phosphoribosyl modification of the ribose at position 64 (21,22,46,58,60). This raises the question of how the vertebrate initiator tRNAs are prevented from acting as elongators in the corresponding protein synthesis systems.…”

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confidence: 99%

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Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Drabkin

Estrella

RajBhandary

1998

Molecular and Cellular Biology

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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 T⌿C stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the T⌿C 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 T⌿C 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.A special methionine tRNA is used for the initiation of protein synthesis in all organisms that have been studied. Of the two classes of methionine tRNAs found universally, the initiator is used exclusively for initiation and the elongator is used for insertion of methionine into internal peptidic linkages (33,48). Eubacteria, mitochondria, and chloroplasts use formylmethionine-tRNA (41) for initiation (10, 33), whereas the cytoplasmic protein synthesis system of eukaryotes uses methionyltRNA (Met-tRNA) without formylation (25, 59).Because of their unique function, initiator tRNAs from eubacteria and eukaryotes possess a number of special properties distinct from those of elongator tRNAs. For eukaryotic cytoplasmic initiator tRNAs, these properties are (i) the formation of a specific complex among the initiator Met-tRNA, eukaryotic initiation factor 2 (eIF2), and GTP (42); (ii) the binding of the initiator Met-tRNA to the ribosomal P site; and (iii) the exclusion of the initiator Met-tRNA from the ribosomal A site. In contrast, elongator aminoacyl-tRNAs form a ternary complex with the eukaryotic elongation factor 1 (eEF1) and GTP and bind to the ribosomal A site.Along with their special properties, eukaryotic initiator tRNAs also possess a number of unique sequence and structural features not found in elongator tRNAs (49, 57). These include (i) an A1:U72...

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confidence: 87%

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confidence: 99%

Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

Drabkin

Estrella

RajBhandary

1998

Molecular and Cellular Biology

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“…We have recently described the use of in vitro labeling in the nucleotide sequence analysis of several eukaryotic cytoplasmic and organellar tRNA species, most of which can neither be conveniently labeled in vivo with [32P] to high specific [1][2][3][4][5][6] activity nor obtained in large quantity . A first step in this work was the development of methods for in vitro lished by partial digestion with snake venom phosphodiesterase, followed by mobility shift analysis using one-dimensional paper C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England electrophoresis 13, or two-dimensional homochromatography4'7,10 of the resulting homologous series of partial digestion products.…”

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confidence: 99%

The use of nuclease P₁in sequence analysis of end group labeled RNA

1977

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A method is described for the direct sequence analysis39f 20-25 nucleotides from the termini of 5'-or 3'-end-group [ P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequences of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.

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“…Evidently tRNAye' has the same nucleotide sequence both in mouse myeloma cells and in rabbit liver. The tRNApet species of these two sources are already known to have identical structures [3]. Fig.…”

Section: Resultsmentioning

confidence: 99%