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: Silke Dorner[show abstract] [hide abstract]
ABSTRACT: The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.Nature Structural & Molecular Biology 12/2004; 11(11):1101-6. · 11.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).Nature Structural & Molecular Biology 06/2006; 13(5):423-8. · 11.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting.Annual Review of Biochemistry 02/2005; 74:129-77. · 27.68 Impact Factor