Minimalist Aminoacylated RNAs as Efficient Substrates for Elongation Factor Tu

Rudinger, Joëlle; Blechschmidt, Bernd; Ribeiro, Sofia; Sprinzl, Mathias

doi:10.1021/bi00185a003

Cited by 49 publications

(50 citation statements)

References 46 publications

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“…reducing translation accuracy) consequently leading to temperature sensitivity. Previous studies have indicated, however, that in vitro tRNAs lacking ψ55 are used in translational elongation with an efficiency indistinguishable from wild-type tRNAs (Nazarenko et al, 1994 ;Rudinger et al, 1994). Measurements of translational accuracy using β-galactosidase thermal lability did not show any indication of misreading occurring in a truB mutant (data not shown), although subtle changes would not be detected with this assay.…”

Section: Discussionmentioning

confidence: 66%

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

et al. 2002

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The truB gene of Escherichia coli encodes the pseudouridine-55 (ψ55) synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. A truB null mutant grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37 to 50 SC. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically inactive allele of truB. The temperature sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding an m 5 U-methyltransferase, modifying the universal U54 tRNA nucleoside, but not by mutations in trmH, encoding the enzyme catalysing the formation of Gm18. The truB mutant proteome contained altered levels of intermediates involved in biogenesis of the outer-membrane proteins OmpA and OmpX. The truB mutation also reduced the basal expression from two σ E promoters, degP and rpoHP3. Three novel aspects to the phenotype of truB mutants were identified. Importantly the data support the hypothesis that TruB-effected ψ55 modification of tRNA is not essential, but contributes to thermal stress tolerance in E. coli, possibly by optimizing the stability of the tRNA population at high temperatures.

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Section: Discussionmentioning

confidence: 66%

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

et al. 2002

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“…Similar stemloops and primary structure are not found in the 5'UTRs of, for example, poliovirus and FMDV. The idea of binding of eIF-2 binding to these sequences in the EMC S'UTR does not conflict with results on minimal requirements of tRNAAsP-binding by EF-Tu [29] and the structural similarities between eIF-2y and EF-Tu [30]. The binding site for EF-Tu in tRNA also resides in the 3' terminal part of the molecule [29].…”

Section: Basepairing Around Aucymentioning

confidence: 89%

Basepairing with 18S ribosomal RNA in internal initiation of translation

Scheper¹,

Voorma²,

Thomas³

1994

FEBS Letters

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In concert with the translation initiation factors 'rrarzs-acting' factors function specifically during internal initiation on picomaviral mRNAs. Of these trans-acting factors, two have been identified as the La-protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18s ribosomal RNA.

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“…The possible importance of tRNA conformation at the end of the acceptor stem and near the junction of the acceptor stem-T⌿C stem extended helix for binding to the EF can be understood from the crystal structure of the T. thermophilus EF-Tu ⅐ GDPNP ⅐ aminoacyl-tRNA ternary complex (44,45) and previous work on the region of the tRNA involved in binding to EF-Tu and eEF1 (27,28,51). With the exception of the 3Ј-terminal A and the aminoacyl-ester group, in the crystal structure, EF-Tu makes no base-specific contacts with the tRNA.…”

Section: Discussionmentioning

confidence: 99%

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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Minimalist Aminoacylated RNAs as Efficient Substrates for Elongation Factor Tu

Cited by 49 publications

References 46 publications

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance

Basepairing with 18S ribosomal RNA in internal initiation of translation

Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

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