Despite the availability of information about the x-ray structure of the ribosome, the molecular basis of dynamic processes within the ribosome during translation remains unknown. To investigate the details of coordinated movement of ribosome components and ligands, we are applying several biochemical approaches.
During the elongation cycle, aminoacyl-tRNA, in a complex with elongation factor Tu (EF-Tu) and GTP, must bind to the ribosome carrying a peptidyl-tRNA at the ribosomal P site and deacylated tRNA at the E site (a site we found to be essential for correct and efficient translocation). EF-Tu binds to the ribosome elongation factor binding center, which is located on the 50S ribosomal subunit on its L7/L12 side. This center consists of two parts: the sarcin-ricin loop (SRL) and the GTPase-associated center (GAC). When the complex of aminoacyl-tRNA–EF-Tu–GTP binds to the ribosome, the anticodon stem-loop of a cognate tRNA must form the codon–anticodon interaction at the decoding center of the ribosome located on the 30S ribosomal subunit. Correct codon–anticodon interaction is thus recognized on the small ribosomal subunit, and the signal must be transmitted to the elongation factor binding center on the large ribosomal subunit, stimulating GTP hydrolysis by EF-Tu, followed by EF-Tu–GDP dissociation from the ribosome. Then the CCA end of aminoacyl-tRNA is accommodated at the peptidyltransferase center (PTC), located at the base of the central protuberance. Only after peptide transfer occurs at the PTC is the ribosome ready to complete the translocation. Elongation factor G, which catalyzes the translocation, must recognize the completeness of the peptidyltransferase reaction, bind to the ribosome, hydrolyze GTP, and stimulate mRNA and tRNA movement. Thus, during the elongation cycle, the signal from either the decoding center or the PTC must reach the elongation factors, which are bound to ribosome binding sites located a significant distance from the other ribosomal functional centers. These signal transmission pathways and the nature of the translocation process are the main interests of the research in our laboratory.
With the help of photoaffinity crosslinking we found, some time ago, that the 5S rRNA D-loop may interact with helix 39 of 23S ribosomal RNA; helix 39 makes direct contact with the elements of the PTC. Mutations in the conserved nucleotide residues of helix 39 result in a number of conformational changes in 23S rRNA within the ribosome. Such changes involve the residues of the PTC and the 5S rRNA D-loop and several located in the helices that connect the PTC with the elongation factor binding center.
Analysis of the structure of the 50S subunit in combination with our data allowed us to identify the helices within 23S rRNA that may form the signal transmission pathway that connects two ribosome functional centers—the PTC and the elongation factor binding site. To test this hypothesis, we inserted mutations into 23S rRNA and the putative signal transmission pathway. Some mutations were lethal, so we developed an approach that allowed us to isolate pure mutant ribosomes. The method is based on the insertion of an RNA apatamer to streptavidin at rRNA elements located on the surface of the ribosome but not involved in its function. Investigation of the functional properties of the mutant ribosomes combined with structure probing confirmed the proposed pathway and revealed new structural elements participating in this signal transmission. We found two elements, which are helices that determine the position of one of the components of the elongation factor binding center—the GAC—relative to the second element—the SRL. We used site-directed mutagenesis to model the relative movement of GAC and found that such movement significantly affects translocation, almost abolishing GTPase activity of EF-G that is stimulated by the appearance of deacylated tRNA at the PTC of the ribosome after peptide transfer but without affecting EF-Tu–related functions. This finding allowed us to hypothesize that elongation factors (EF-Tu or EF-G) that bind to the ribosome only at precisely defined steps of the elongation cycle recognize different conformations of the ribosome; this occurs when the mutual orientation of the two elements of the elongation factor binding center is favorable for only one of the factors, causing sequential binding of the factors during the elongation cycle. Moreover, as part of its function, one of the factors changes the ribosome conformation in such a way as to prepare the proper binding site for the next elongation factor.
Translocation remains one of the most mysterious processes the ribosome performs. Deacylated tRNA appearing at the ribosome in the course of peptide transfer stimulates GTPase hydrolysis on EF-G. Translocation first happens on the 50S ribosomal subunit, and hybrid P/E and A/P sites are formed as translocation intermediates. To investigate the importance of these translocation intermediates for signal transmission from the PTC to EF-G, we created ribosomes with a mutation in 23S rRNA that significantly destabilized the interaction of the CCA end of deacylated tRNA with the ribosome at the E site. Investigation of the properties of this mutant allowed us to determine that formation of the P/E hybrid state is important for precise translocation and significantly affects the stimulation of GTP hydrolysis by EF-G. These data allowed us to suggest that initial binding of EF-G produces a conformation signal that goes to the PTC and stimulates movement of the CCA end of deacylated tRNA to the E site on the 50S ribosomal subunit; in turn, formation of the transitional empty P loop at the PTC stimulates transmission of the conformational signal to the EF-G binding site, causing activation of EF-G GTPase, which is necessary for further translocation on the 30S subunit.
Many modified bases in rRNA are conserved. Some modifications are known to be essential for antibiotic resistance, but the function of conserved modifications is not clear. To investigate this problem, we identified several RNA methyltransferases that modify a particular RNA base at different steps of ribosome assembly.
Transfer-messenger RNA (tmRNA) rescues stalled ribosomes by switching translation from cellular mRNA to tmRNA. We investigated tmRNA structure and proposed a hypothesis to explain tmRNA passage through the ribosome. To test this hypothesis, we developed an approach that allowed us to isolate tmRNA–ribosomal complexes blocked at defined steps of the translation process and obtained experimental data in support of our concepts.
Last updated September 2008