Structural Insights into Ribosome Recycling Factor Interactions with the 70S Ribosome

Pai, R.D.; Zhang, Wen; Schuwirth, B.S.; Hirokawa, Go; Kaji, Hideko; Kaji, Akira; Cate, J.H.D.

doi:10.1016/j.jmb.2007.12.048

Cited by 53 publications

(57 citation statements)

References 53 publications

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“…Recent studies have shown that ribosomes isolated from a strain lacking RluD show defects in the ability of RF2 (but not RF1) to catalyze the release of a peptide on UAA-programmed ribosome complexes (14). Finally, other studies observed large conformational changes in H69 in ribosome structures where ribosome recycling factor is bound, consistent with the idea that movement of the helix plays a critical role in the dissociation reaction that ribosome recycling factor catalyzes (9). Simple positioning makes H69 an excellent candidate for participation in substrate selection events and overall ribosome function.…”

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confidence: 61%

“…Structural studies of tRNA (2)(3)(4)(5), release factors (6 -8), and ribosome recycling factor (9,10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).…”

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confidence: 99%

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Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

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confidence: 61%

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Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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“…The interaction of RRF with the ribosome has been studied by directed hydroxyl radical footprinting on ribosomal RNA (Lancaster et al 2002), by cryo-EM (Agrawal et al 2004;Gao et al 2005;Barat et al 2007;Gao et al 2007), and by X-ray crystallography (Wilson et al 2005;Borovinskaya et al 2007;Weixlbaumer et al 2007;Pai et al 2008). RRF is bound at the interface between the subunits where it interacts mainly with the 50S subunit.…”

Section: Ribosome Disassembly As An In Vivo Target Of Fusidic Acidmentioning

confidence: 99%

Distinct functions of elongation factor G in ribosome recycling and translocation

Savelsbergh

Rodnina

Wintermeyer

2009

RNA

117

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Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.

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“…23 In the cocrystal structures of ribosomes with various translation factors, bridge B2a has been seen interacting with parts of IF2, eukaryotic translation elongation factor 2, release factor 2, release factor 3 and ribosome recycling factor. [24][25][26][27][28][29][30] These interactions suggest that bridge B2a could be involved in regulating the activity of the translation factors when they are bound to the 70S ribosome during protein synthesis. One of the best-characterized interactions of B2a is with translation termination factors.…”

Section: Introductionmentioning

confidence: 99%