Role of ribosomal protein L27 in peptidyl transfer
ABSTRACT The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.
- SourceAvailable from: Erin Ashley Wall
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- "It is not clear whether L27 does get cleaved, but if so, the same protease is likely responsible for the cleavage of gp46 and gp47 as well. L27 has an unusual structure with a long N-terminal extension that reaches into the peptidyl transfer site of the ribosome (Wang et al., 2004; Trobro and Aqvist, 2008), and is a target for several antibiotics (Sonenberg et al., 1973; Tejedor and Ballesta, 1986; Colca et al., 2003). It is not unlikely that the N-terminal extension in S. aureus L27 affects ribosome function and that its cleavage would be an essential process. "
ABSTRACT: 80α is a temperate, double-stranded DNA bacteriophage of Staphylococcus aureus that can act as a "helper" for the mobilization of S. aureus pathogenicity islands (SaPIs), including SaPI1. When SaPI1 is mobilized by 80α, the SaPI genomes are packaged into capsids that are composed of phage proteins, but that are smaller than those normally formed by the phage. This size determination is dependent on SaPI1 proteins CpmA and CpmB. Here, we show that co-expression of the 80α capsid and scaffolding proteins in S. aureus, but not in E. coli, leads to the formation of procapsid-related structures, suggesting that a host co-factor is required for assembly. The capsid and scaffolding proteins also undergo normal N-terminal processing upon expression in S. aureus, implicating a host protease. We also find that SaPI1 proteins CpmA and CpmB promote the formation of small capsids upon co-expression with 80α capsid and scaffolding proteins in S. aureus.Virology 09/2012; 434(2). DOI:10.1016/j.virol.2012.08.031 · 3.28 Impact Factor
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- "The recent advances in ribosome crystallography at nearatomic resolution (Selmer et al, 2006; Laurberg et al, 2008; Weixlbaumer et al, 2008; Gao et al, 2009; Schmeing et al, 2009; Schuette et al, 2009; Voorhees et al, 2009) in combination with powerful molecular simulation techniques (Carlsson et al, 2008; Trobro and Aqvist, 2008; Wallin and Aqvist, 2010) may make such a quantitative structural analysis feasible in the near future. In fact, the power of such a combination has recently been demonstrated by the explanation (Trobro and Aqvist, 2009; Sund et al, 2010) of the accuracy of codon reading by class-1 release factors (Freistroffer et al, 2000). "
ABSTRACT: We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.The EMBO Journal 01/2011; 30(2):289-301. DOI:10.1038/emboj.2010.328 · 10.75 Impact Factor
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- "Moreover, recent crystallographic data on the full 70S ribosome from Thermus thermophilus  show that ribosomal protein L27 extends with its N-terminus into the PTC and makes contact with the tRNA substrates. Finally, Trobro and Aqvist have investigated the role of L27 in peptidyl transfer by carrying out computer simulations, employing the 2.8 Å T. thermophilus structure as starting coordinates . "
ABSTRACT: Periodate-oxidized tRNA (tRNAox), the 2',3'-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3'-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA(Asp)ox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA(Asp) competed with tRNA(Asp)ox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.Biochimie 08/2009; 91(11-12):1420-5. DOI:10.1016/j.biochi.2009.07.013 · 3.12 Impact Factor