Role of the 1-72 base pair in tRNAs for the activity of<i>Escherichia coli</i>peptidyl-tRNA hydrolase

Dutka, Sophie; Meinnel, Thierry; Lazennec, Christine; Mechulam, Yves; Blanquet, Sylvain

doi:10.1093/nar/21.17.4025

Cited by 61 publications

(43 citation statements)

References 20 publications

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“…The fold-back conformation could also contribute to discrimination of the initiator tRNA from elongator tRNAs by other proteins such as the elongation factor EF-Tu and peptidyl-tRNA hydrolase (4,6,(30)(31)(32)(33) (4,35).…”

Section: Discussionmentioning

confidence: 99%

“…Many proteins, including aminoacyl-tRNA synthetases and RNA enzymes such as the Ml RNA component of RNase P, utilize the acceptor stem and 3' end of tRNA for binding discrimination (3,4,6,30,33,34,(38)(39)(40)(41)(42)(43)(44). Therefore, it is important to consider in all ofthese cases the contribution of3'-terminal conformation on specific recognition of tRNAs.…”

Section: Discussionmentioning

confidence: 99%

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NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.

Puglisi

Williamson

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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An important step in initiation of protein synthesis in Escherichia col is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The deterinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelces, which correspond to the acceptor stem of mutants of E. coUi initator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs.One of the mutant tRNAs is an extremely poor substrate for Met-tRNA trausformylase, whereas the other one is a much better substrate. We show that one microhelix forms a struchire in which its 3'-ACCA sequence extends the tacklng of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in dose proximity to GI. These results highiight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA trandormylase.Protein synthesis in Escherichia coli is initiated with formylmethionyl-tRNA (tMet-tRNA). A crucial step in this process is the specific formylation of methionine attached to tRNAfmet by Met-tRNA transformylase (EC 2.1.2.9) (1, 2). We showed previously that the major determinants for formylation are clustered in the acceptor stem of tRNA (3). One of these determinants is a mismatch (as found in tRNAfmet) or a weak base pair at the end of the acceptor stem. tRNAs carrying the wild-type ClxA72 or virtually any other mismatch are good substrates, whereas those carrying stable base pairs such as ClG72 or GlFC72 are extremely poor substrates (4-6). These results suggest a requirement for nucleotides 1 and 72 to be unpaired during formylation.The strong negative effect of ClG72 or G1C72 base pairs on formylation can be compensated for by an additional mutation of A73, the discriminator base, which precedes the CCA sequence common to all tRNAs, to a pyrimidine such as U73 (4). For example, in contrast to the ClG72 mutant, which is a very poor substrate (V 1/K aPP down by a factor of495 compared to wild-type tRNA), the ClG72/U73 mutant is almost as good a substrate as wild-type tRNA (V./KIPP down only by a factor of 3.7). Similarly, compared to the G1*C72 mutant, the G1'C72/U73 mutant is a better substrate for Met-tRNA transformylase (V./KIPP down by a factor of 60, while the factor is 1035 for the G1-C72 mutant). On the basis of these results, we proposed that the discriminator base influences the stability of the terminal base pair in the acceptor stem and/or structure of tRNA at the 3' end (4, 6).In this paper, we use NMR spectroscopy (7) to study the effect of the discriminator base (8) on tRNA conformation at the 3' end. Two RNA oligonucleotides that correspond to variants of the acceptor stem of initiator tRNAfmet were designed for NMR study (Fig. 1A). These oligonucleotides contain the seven ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.

Puglisi

Williamson

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…1.29) is a well-characterized cytoplasmic enzyme that cleaves peptidyl-tRNA or N-acyl-aminoacyl-tRNA to yield free peptides of N-acyl-amino acid and tRNA; its natural substrates probably are peptidyl-tRNAs that drop off the ribosome during pro- tein synthesis (Garcia-Villegas et al, 1991;Meinnel et al, 1993, and references therein). PTH is a rare enzyme with about 25 molecules per cell (Dutka et al, 1993 The significance of this similarity is strongly supported by the conservation pattern in a multiple alignment of several other prokaryotic proteins related to PTH (Fig. 2A).…”

Section: Resultsmentioning

confidence: 71%

New protein functions in yeast chromosome VIII

et al. 1995

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(Johnston et al., 1994) reported the function of 46% of the probable 269 gene products, either as experimentally known or as predicted by exploiting evolutionary relationships to proteins of known function.As of the submission date of this paper, we have been able to add functional information to another 9% of the putative ORFs, by searching more recent versions of sequence databases and using improved similarity search tools (Bork et al., 1992;Koonin et al., 1994;Scharf et al., 1994). As a result, the level of tentative functional assignment for chromosome VI11 is now above 50% of all ORFs (Fig. 1). Results and discussion:The newly identified tentative functions range from enzymes (e.g., chelate synthase, glutaminase-asparaginase, gamma-butyrobetaine hydroxylase, 2-deoxyglucose-' Present address:

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“…A method for the purification of E. coli Pth has been reported previously (Dutka et al, 1993;; however, we purified this enzyme more easily and effectively with a His tag as follows. The gene encoding E. coli Pth was PCR-amplified and cloned into the pET-15b vector (Novagen).…”

Section: Overexpression and Purification Of E Coli Pthmentioning

confidence: 99%

“…Indeed, formylated initiator tRNA must be kept intact in order to participate in the formation of the ribosomal initiator complex. This resistance is thought to be a consequence of the lack of base pairing at position 1-72 in tRNA f Met , which may cause incorrect positioning of the 5 0 -phospate that is necessary for Pth activity (Schulman & Pelka, 1975;Dutka et al, 1993). However, the molecular mechanism of this substrate discrimination remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase fromEscherichia coliin complex with the acceptor-TΨC domain of tRNA

et al. 2011

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Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the product of aborted translation. In the present work, Pth from Escherichia coli was crystallized with the acceptor-TÉC domain of tRNA using 1,4-butanediol as a precipitant. The crystals belonged to the hexagonal space group P6 1 , with unit-cell parameters a = b = 55.1, c = 413.1 Å , and diffracted X-rays beyond 2.4 Å resolution. The asymmetric unit is expected to contain two complexes of Pth and the acceptor-TÉC domain of tRNA (V M = 2.8 Å 3 Da À1 ), with a solvent content of 60.8%. The structure is being solved by molecular replacement.

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Role of the 1-72 base pair in tRNAs for the activity ofEscherichia colipeptidyl-tRNA hydrolase

Cited by 61 publications

References 20 publications

NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.

NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.

New protein functions in yeast chromosome VIII

Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase fromEscherichia coliin complex with the acceptor-TΨC domain of tRNA

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