Role of Ribosomal Protein L27 in Peptidyl Transfer †
Department of Cell and Molecular Biology, Uppsala Biomedical Center, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden. Biochemistry
(Impact Factor: 3.02).
05/2008; 47(17):4898-906. DOI: 10.1021/bi8001874
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.
Available from: Ingo Wohlgemuth
- "When Pmn is used as substrate, the peptidyl-transfer (PT) reaction shows a pronounced pH dependence with two ionizing groups, indicating that besides the amino group of the substrate (6.9 for Pmn [Katunin et al. 2002]), another ionizing group, with a pKa of ∼7.5, contributes to catalysis (Katunin et al. 2002; Beringer and Rodnina 2007). Based on computer simulations of the pre-and post-PT state of the ribosome, this group has been proposed to be the N-terminal amine of L27 (Trobro and Åqvist 2008; Xiao and Wang 2012). The absence of L27 is thus predicted to alter the pH dependence of PT; however, the contribution of this ionizable group to the PT reaction has not been experimentally tested so far. "
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ABSTRACT: 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 puromy
Available from: PubMed Central
- "Furthermore, one may speculate that the ribosomal machinery is slowed down in a rate-limiting step by changes connected with the partial replacement of H-atoms by deuterons in this multisubunit protein complex. The importance of water and its hydrogen-bonding networks for ribosomal function was reported by  which represents a major factor of the ribosomal entropic stabilization [35–37]. "
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ABSTRACT: The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.
Available from: Erin Ashley Wall
- "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. "
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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.
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