Contribution of ribosomal residues to P-site tRNA binding (original) (raw)
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Direct tRNA-protein interactions in ribosomal complexes
Nucleic Acids Research, 1991
Nucleotide residues in E. coil tRNAPhe interacting directly with proteins in pre-and posttranslocated ribosomal complexes have been identified by UVinduced cross-linking. In the tRNAPIW molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNAPhe located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex.
Different conformations of tRNA in the ribosomal P-site and A-site
European journal of biochemistry / FEBS, 1985
Footprinting studies involving radioactively end-labelled tRNA species bound at either the ribosomal P- or A-site have yielded information that the tRNA's conformation is different in the two sites. Appropriate controls showed the relevance of using poly(U)-directed tRNAPhe binding in the P-site and Phe-tRNAPhe in the A-site. Digestion of the tRNA species was effected by RNases T1, T2 and cobra venom RNase. Experiments were performed with tRNAs 32P-labelled at either end to establish positions of primary cuts more confidently. In addition to the common protection of the aminoacyl-stem and anticodon-arm, footprinting experiments revealed striking differences in the accessibility of the T- and D-loops of tRNAs bound in the P- and A-sites. We observed a more open structure for the tRNA in the A-site. These results are consistent with a dynamic structure of tRNA during the translocation step of protein biosynthesis.
Proceedings of the National Academy of Sciences, 1985
A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome (tRNA cross-linking/aminacyl site/peptidyl site/transladonal fidelity/kfrromycin) ABSTRACT Previously, we reported that the antibiotic kirromycin induces two tRNA-binding sites on the elongation factor Tu. The classical binding site (site I) binds aminoacyl-tRNA and, with much less affinity, deacylated tRNA. The kirromycin-induced site II binds aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA with comparable affinities. Accordingly, 3'-oxidized tRNA can be cross-linked in the presence of the antibiotic to two specific sites of EF-Tu: Lvs-237 and Lys-208. Here, we report that 3'-oxidized tRNAh, bound to a ribosome-poly(U) complex, can also be crosslinked to either one of these two sites. When located in the ribosomal peptidyl site, it cross-links exclusively to Lys-208; when located in the ribosomal aminoacyl site, it cross-links exclusively to Lys-237, irrespective of the presence of kirromycin. Since no cross-linking could be detected in the absence of ribosomes and kirromycin, we conclude that the tRNA-binding site II is induced upon interaction of aminoacyl-tRNA-EF-Tu-GTP with the ribosome-mRNA complex. The results indicate that, on the ribosome, EF-Tu interacts with peptidyl-sitebound peptidyl-tRNA through tRNA-binding site II and with aminoacyl-site-bound aminoacyl-tRNA through tRNA-binding site I.
FEBS Letters, 2002
Photoreactive derivatives of yeast tRNA Phe containing 2-azidoadenosine at their 3P P termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA Phe , Phe-tRNA Phe and N-acetyl-Phe-tRNA Phe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA Phe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA Phe probe also labeled protein L1. Crosslinking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit. ß
Journal of Biological Chemistry, 2000
Photoreactive derivatives of yeast tRNA Phe containing 2-azidoadenosine at their 3P P termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA Phe , Phe-tRNA Phe and N-acetyl-Phe-tRNA Phe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA Phe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA Phe probe also labeled protein L1. Crosslinking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit. ß
Specificity of the ribosomal A site for aminoacyl-tRNAs
Nucleic Acids Research, 2008
Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotidesubstituted mRNAs are used to demonstrate the presence of a lower threshold in k off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.
A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding
Nucleic Acids Research, 2010
High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 'P-site loop'. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.