A Conserved Base-pair between tRNA and 23 S rRNA in the Peptidyl Transferase Center Is Important for Peptide Release

Feinberg, Jason S.; Joseph, Simpson

doi:10.1016/j.jmb.2006.09.040

Cited by 23 publications

(16 citation statements)

References 51 publications

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“…Compared to the abasic 2602 ribosome, the C3-linker containing PTC showed at least 50-fold slower peptidyl-tRNA hydrolysis rates. Peptidyl-tRNA-binding deficiencies to the P-site in the 2602 C3-linker ribosome are unlikely since essentially wild-type levels of peptidyl transfer activities remained (Figure 3C) and none of the interactions of P-tRNA with the 23S rRNA that have been identified recently to be important for efficient peptide release (46), have been manipulated. Applying an RF-independent peptidyl-tRNA hydrolysis assay which was activated by the A-site-bound 3′-terminal CCA end of deacylated tRNA, again revealed a significantly decreased activity when the C3-linker modification was introduced at position 2602 (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Amort¹,

Wotzel²,

Bąkowska-Żywicka³

et al. 2007

Nucleic Acids Research

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Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2′-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.

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Section: Discussionmentioning

confidence: 99%

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Amort¹,

Wotzel²,

Bąkowska-Żywicka³

et al. 2007

Nucleic Acids Research

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“…Time courses for peptide release were carried out using 0.25 μM release complexes and 20 μM RF1 (both wild type and H197A). Each time point was quenched using 25% formic acid, run on an eTLC plate, and analyzed as described previously (15). Peptide release experiments were repeated four times.…”

Section: Methodsmentioning

confidence: 99%

Histidine 197 in Release Factor 1 Is Essential for A Site Binding and Peptide Release

et al. 2010

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Class I peptide release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNAprotein interface that appear critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (197 in E. coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1•ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue. KeywordsRibosome; release factor; stop codon; translation; termination The entry of a stop codon into the ribosomal A site signals the termination phase of protein synthesis. The nearly universally conserved stop codons UAA, UAG, and UGA are recognized by class I release factors (RFs) (1). In bacteria, there are two class I release factors: RF1 and RF2. RF1 recognizes UAA and UAG, while RF2 recognizes UAA and UGA (2). In eukaryotes, a single class I release factor, eRF1, recognizes all three stop codons (3). Following the recognition of the stop codon in the A site, class I release factors trigger peptidyl-tRNA hydrolysis and the release of the newly synthesized protein from the ribosome (4). Accurate recognition of stop codons by RFs is essential to prevent premature termination, which would be costly to the cell. The fidelity of stop codon recognition by RFs has been estimated to be 1 x 10 −3 to 1 x 10 −6 (5,6), which is arguably more accurate than tRNA selection on cognate codons. Remarkably, this high level of accuracy in stop codon recognition is achieved by RFs without the help of a proofreading mechanism.Previously, the kinetics of RF1 and RF2 discrimination between stop and sense codons were systematically analyzed under steady state conditions (6). These studies showed that a sense codon in the decoding center increases the K M between the release factors and the ribosome by 400-3000-fold, while the catalytic rate constant for peptide release (k cat ) was reduced by More recently, the binding of RF1 to ribosomes with stop or sense codons in the decoding center was directly measured using a fluorescence-based, pre-steady state kinetic assay (7). These studies showed that the association rate constant of RF1 with the ribosome is similar with both sense and stop codons, while t...

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“…1; Samaha et al 1995;Kim and Green 1999). While the specific interactions of tRNAs in classical positions are critical for both peptide bond formation (Lieberman and Dahlberg 1994;Weinger et al 2004;Brunelle et al 2006;Dorner et al 2006) as well as peptide release (Feinberg and Joseph 2006), such contacts must be extensively remodeled for tRNA substrates to move directionally through the ribosome during active translation (Agirrezabala and Frank 2009;Munro et al 2009). …”

Section: Introductionmentioning

confidence: 99%

Insights into the molecular determinants of EF-G catalyzed translocation

Wang¹,

Altman

Blanchard

2011

RNA

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Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 39-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 39-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.

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A Conserved Base-pair between tRNA and 23 S rRNA in the Peptidyl Transferase Center Is Important for Peptide Release

Cited by 23 publications

References 51 publications

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Histidine 197 in Release Factor 1 Is Essential for A Site Binding and Peptide Release

Insights into the molecular determinants of EF-G catalyzed translocation

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