Ribosomes and Protein Synthesis
Wednesday, April 1, 2009
Presented by the Chemical Biology Discussion Group
Recent years have seen an increasing level of dialogue between chemists and biologists, the lines of communication consolidated by the availability of recombinant biotechnology tools for manipulating the chemical structure of genes, and the proteins they encode. This has led to an explosion of interdisciplinary activity at the chemistry/biology interface, now coined chemical biology. The Chemical Biology Discussion Group brings together chemists and biologists interested in hearing about the latest ideas in this rapidly growing field. Meetings of this group will provide a forum for lively discussion and for establishing connections, and perhaps collaborations, between chemists armed with novel technologies and biologists receptive to using these approaches to solve their chosen biological problem.
Dealing with Mistakes: The Ribosome's Response to Misincorporations
Hani Zaher, The Johns Hopkins University School of Medicine
Accurate translation of the genetic information into peptide sequences is a defining feature of the ribosome and the translation factors. While much is known about the events that lead to ribosomal discrimination against the incorporation of the wrong amino acid, little is known about the ribosomal response following a miscoding event. Using a well-defined in vitro bacterial translation system, we have systematically evaluated the effect of codon:anticodon mismatches in the P and E sites on the selectivity of the A site towards release factors and aminoacylated tRNAs. Our results argue that following a misincorporation during protein synthesis, the A site of the ribosome becomes generally promiscuous (or ram) leading ultimately to premature release of defective polypeptide chains. The dramatic effects that we observe argue that the ribosome, like DNA polymerase, depends on a proofreading mechanism following peptide bond formation to ensure the high fidelity of protein synthesis observed in vivo. We suggest that such a system may play even more important roles in the cell under conditions of amino acid starvation and cellular stress and may help to explain the stringent control of RF2 expression in bacteria.
Coupling of Ribosome and tRNA Dynamics during Translation
Ruben Gonzalez, Columbia University
Comparisons of X-ray crystallographic and cryogenic electron microscopic structures of ribosomal complexes have led to the hypothesis that conformational dynamics of the ribosome, its transfer RNA (tRNA) substrates, and associated translation factors play important mechanistic and regulatory roles throughout all stages of protein synthesis. Using fluorescently-labeled components within a highly-purified in vitro translation system, we are directly characterizing structural changes of the translational machinery in real time using single-molecule Förster resonance energy transfer (smFRET) in order to elucidate the mechanisms through which these dynamics direct and regulate the individual steps of translation. Here we report smFRET signals that have allowed us to fully characterize the intrinsic conformational dynamics of a ribosomal domain, the L1 stalk, as well as the coupling between L1 stalk and tRNA dynamics, throughout protein synthesis. Our data reveal that the translating ribosome can spontaneously and reversibly fluctuate between two global conformational states, and that transitions between these two states involve coupled movements of the L1 stalk and ribosome-bound tRNAs, accompanied by ratcheting of the ribosomal subunits. Furthermore, we find that translation factors uniquely recognize these global states of the ribosome and differentially affect transition rates between the two states. Thus, translation factor-mediated recognition and control of intrinsic ribosome dynamics plays a major mechanistic role during translation. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of translation factors and a unifying theme throughout protein synthesis.
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