Activities of the peptidyl transferase center of ribosomes lacking protein L27

Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V.

doi:10.1261/rna.053330.115

Cited by 19 publications

(19 citation statements)

References 36 publications

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“…Deletion of three N-terminal amino acids of L27 inhibits cell growth and impairs peptidyl-transferase activity [26]. Recent work, however, argues against the involvement of L27 in the peptidyl-transferase activity, as ΔL27 ribosomes were found to catalyze peptidyl transfer similarly to wild-type ribosomes in vitro [27]. Because the peptidyl-transferase centers of archaeal and eukaryotic ribosomes do not contain an L27 homolog and the mechanism of peptidyl transfer is universally conserved, bacterial L27 is unlikely to be critical for peptidyl transfer.…”

Section: Resultsmentioning

confidence: 99%

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Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

Svidritskiy

Madireddy

Коростелев

2016

Journal of Molecular Biology

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Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of E. coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial translation termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of termination.

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Section: Resultsmentioning

confidence: 99%

“…A recent study reports that a saturating concentration (1.5 μM) of RF2 catalyzes peptide release at nearly identical rates from wild-type and ΔL27 ribosomes [27], suggesting that L27 does not modulate the catalytic activity of release factors. Instead, L27 may regulate termination efficiency by modulating the affinity of release factors.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

Svidritskiy

Madireddy

Коростелев

2016

Journal of Molecular Biology

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“…In accordance with these structural data, Polikanov et al (5), have recently proposed that the N-terminus of T. thermophilus L27 might take part in peptide bond formation. In sharp contrast with these structural data, it was recently demonstrated that L27 doesn't play any significant role in peptide-bond formation (6). Moreover, ribosomes from archaea or eukaryotes do not have protein L27 or any homologous counterpart, indicating that L27 cannot be a part of an evolutionarily conserved peptidyl transfer mechanism.…”

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confidence: 85%

Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative

Hountondji

Créchet

Caër

et al. 2017

The Journal of Biochemistry

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In this report, we have used periodate-oxidized tRNA (tRNAox) as an affinity laleling reagent to demonstrate that: (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribosomes; (ii) the stoichiometry of labelling is one molecule of tRNAox bound to one polypeptide chain of endogenous bL12; (iii) cross-linking in situ of bL12 with tRNAox on the ribosomes provokes the loss of activity; (iv) intact tRNA protects bL12 in the 70S ribosomes against cross-linking with tRNAox; (v) both tRNAox and pyridoxal 5'-phosphate (PLP) compete for the same or for proximal cross-linking site(s) on bL12 inside the ribosome; (vi) the stoichiometry of cross-linking of PLP to the recombinant E. coli bL12 protein is one molecule of PLP covalently bound per polypeptide chain; (vii) the amino acid residue of recombinant bL12 cross-linked with PLP is Lys-65; (viii) Lys-65 of E. coli bL12 corresponds to Lys-53 of eL42 which was previously shown to cross-link with P-site bound tRNAox on human 80S ribosomes in situ; (ix) finally, E. coli bL12 and human eL42 proteins display significant primary structure similarities, which argues for evolutionary conservation of these two proteins located at the tRNA-CCA binding site on eubacterial and eukaryal ribosomes.

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“…However, a recent study of the impact of L27 on the activity of PTC argues against a key role of L27 in peptide bond formation on the ribosome, although the lack of L27 slightly reduced the average elongation rate in vitro (9). This implies that the slow-growth phenotype of the L27-deficient strain may be attributed to the impaired ribosome assembly, reducing the pool of active ribosomes inside the cell, rather than to defects in the PTC (9). As for the rpmA-encoded L21, it has been reported to interact with 23S rRNA (10), but we still do not know much about its functional importance.…”

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confidence: 99%

“…The L27-lacking strain has severe growth defects and is deficient in both the assembly and functioning of the 50S ribosomal subunits (7). However, a recent study of the impact of L27 on the activity of PTC argues against a key role of L27 in peptide bond formation on the ribosome, although the lack of L27 slightly reduced the average elongation rate in vitro (9). This implies that the slow-growth phenotype of the L27-deficient strain may be attributed to the impaired ribosome assembly, reducing the pool of active ribosomes inside the cell, rather than to defects in the PTC (9).…”

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confidence: 99%

Regulation of Ribosomal Protein Operons rplM-rpsI , rpmB-rpmG , and rplU-rpmA at the Transcriptional and Translational Levels

2016

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It is widely assumed that in the best-characterized model bacterium Escherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several E. coli r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the in vivo regulation of three r-protein operons, rplM-rpsI, rpmB-rpmG, and rplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal lacZ gene, we show here that at the translation level only one of these operons, rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5= untranslated region (UTR) of the operon mRNA, while rpmB-rpmG and rplU-rpmA are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of E. coli r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of rplM-rpsI expression in vivo, acting at a target within the rplM translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria. IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents. Ribosomal protein (r-protein) operons rplM-rpsI, rplU-rpmA, and rpmB-rpmG encode r-proteins L13-S9, L21-L27, and L28-L33, respectively. Despite long-term studies of the regulation of expression of ribosomal components (1-5), these three operons have never been explored and their regulation remains largely unexplained. At the same time, the products of the operons play noticeable roles in ribosome assembly and functioning, which stimulates investigation into the control mechanisms of their expression, given the importance of knowledge about ribosome biogenesis and its control.Proteins L21 and L27 encoded by rplU-rpmA are bacterium specific (6). L27 was suggested to contribute to the function of the ribosomal peptidyl transferase center (PTC) via interactions of its N-terminal tail with both the A-site and P-site tRNAs, facilitating their accommodation in the PTC (7, 8). The L27-lacking strain has severe growth defects and is deficient in both the assembly and funct...

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Activities of the peptidyl transferase center of ribosomes lacking protein L27

Cited by 19 publications

References 36 publications

Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

Affinity labelling in situ of the bL12 protein on E. coli 70S ribosomes by means of a tRNA dialdehyde derivative

Regulation of Ribosomal Protein Operons rplM-rpsI , rpmB-rpmG , and rplU-rpmA at the Transcriptional and Translational Levels

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