1,720,972 research outputs found
Activities of the peptidyl transferase center of ribosomes lacking protein L27.
Full text linkThe 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
Studying macromolecular complex stoichiometries by peptide-based mass spectrometry.
No full textA majority of cellular functions are carried out by macromolecular complexes. A host of biochemical and spectroscopic methods exists to characterize especially protein/protein complexes, however there has been a lack of a universal method to determine protein stoichiometries. Peptide-based MS, especially as a complementary method to the MS analysis of intact protein complexes, has now been developed to a point where it can be employed to assay protein stoichiometries in a routine manner. While the experimental demands are still significant, peptide-based MS has been successfully applied to analyze stoichiometries for a variety of protein complexes from very different biological backgrounds. In this review, we discuss the requirements especially for targeted MS acquisition strategies to be used in this context, with a special focus on the interconnected experimental aspects of sample preparation, protein digestion, and peptide stability. In addition, different strategies for the introduction of quantitative peptide standards and their suitability for different scenarios are compared
Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates.
Full text linkThe ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNAPhe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was similar to 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation
Optimization of speed and accuracy of decoding in translation.
Full text linkThe speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10(-3), consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k(cat) values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near-and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation. The EMBO Journal (2010) 29, 3701-3709. doi:10.1038/emboj.2010.229; Published online 14 September 201
Rapid peptide bond formation on isolated 50S ribosomal subunits.
Full text linkThe catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre
Translation error clusters induced by aminoglycoside antibiotics
No full textAbstract Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs
Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes
No full textThe emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for >1,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Essential structural elements in tRNAPro for EF-P-mediated alleviation of translation stalling.
Full text linkThe ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNAPro affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNAPro is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNAPro isoacceptors and tRNAfMet in Escherichia coli, and the D-arm of tRNAfMet is essential for EF-P-induced acceleration of fMet–puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm
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