Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

Wower, Iwona K.; Wower, Jacek; Zimmermann, Robert A.

doi:10.1074/jbc.273.31.19847

Cited by 65 publications

(73 citation statements)

References 42 publications

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“…Thus, incorporation of L16 possibly triggers a significant conformational change and facilitates organization of the architecture of the functional site in the 50 S subunit (48). On the other hand, deletion of rpmA encoding L27 in E. coli results in accumulation of the premature 40 S subunit lacking L16, L20, L21, and L27 and inhibition of the peptidyl transferase activity of 70 S ribosome (49). Incorporation of L16 into the 50 S subunit depends on L25, which additionally interacts with YlqF, as evident from the E. coli 50 S subunit assembly map (50).…”

Section: Discussionmentioning

confidence: 99%

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Matsuo

Morimoto²,

Kuwano

et al. 2006

Journal of Biological Chemistry

111

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Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.

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Section: Discussionmentioning

confidence: 99%

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Matsuo

Morimoto²,

Kuwano

et al. 2006

Journal of Biological Chemistry

111

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“…Escherichia coli strains in which L27 was deleted grew six times slower than the wild type (wt), and ribosomes lacking L27 were found to carry sub-stoichiometric amounts of L16, L21, and L20. The PT activity of ΔL27 ribosomes was slightly reduced, both in the presence of Pmn or native A-site substrate (Phe-tRNA Phe ) (Wower et al 1998). Truncation of the N-terminal tail of L27 revealed that the absence of as few as the first three residues reduces the PT activity to the level of ΔL27 ribosomes (Maguire et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Activities of the peptidyl transferase center of ribosomes lacking protein L27

Maracci

Wohlgemuth

Rodnina

2015

RNA

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The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A-and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally.Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

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“…Several ribosomal proteins of the small subunit (17)(18)(19)(20) and at least one of the large subunit (21) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22)(23)(24).…”

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confidence: 99%

A Dispensable Yeast Ribosomal Protein Optimizes Peptidyltransferase Activity and Affects Translocation

Dresios

Panopoulos

Suzuki³

et al. 2003

Journal of Biological Chemistry

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Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a secondorder rate constant (k cat /K s ) corresponding to the rate of peptide bond formation; this k cat /K s was lowered 3-fold to 1.15 min ؊1 mM ؊1 in the L41 mutant compared with 3.46 min ؊1 mM ؊1 in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A-to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 M EF2 compared with 0.02 M for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 M of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 M in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.The ribosome is a large macromolecular machine that consists of a large number of proteins and several molecules of ribosomal RNA (rRNA). The ribosome is responsible for the translation of the genetic message, which results in protein synthesis.The crystal structures of the 30 S (1, 2) and 50 S (3, 4) ribosomal subunits, and the intact 70 S ribosome (5) are contributing to a better understanding of ribosomal function. It has now been confirmed that rRNA plays the major role in ribosomal structure and function, including the two most important activities of the ribosome: the decoding process and the peptidyltransferase activity. The 16 S rRNA of the small ribosomal subunit is responsible for the decoding process, the selection of the cognate tRNA (6). The central loop of domain V of 23 S rRNA of the large ribosomal subunit is responsible for the catalytic activity of the ribosome (7, 8). The search is still underway to identify a critical site or nucleotide(s) of 23 S rRNA involved in the catalysis of peptide bond formation (9 -13). It is still possible that the rate of peptide bond formation is influenced by structural rearrangements of RNA in the peptidyltransferase center that would affect the positioning of the substrates (14).Ribosomal proteins offer structural support to the ribosome by stabilizing and orienting the ribosomal RNA into a specific, active structure (15). Also, they are crucial for the assembly of functional ribosomes (16). Several ribosomal proteins of the small subunit (17-20) and at least one of the large subunit (21) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22-24).Yeast ribosomal protein L41 is the smallest a...

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Ribosomal Protein L27 Participates in both 50 S Subunit Assembly and the Peptidyl Transferase Reaction

Cited by 65 publications

References 42 publications

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

Activities of the peptidyl transferase center of ribosomes lacking protein L27

A Dispensable Yeast Ribosomal Protein Optimizes Peptidyltransferase Activity and Affects Translocation

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