Mechanistic Studies of Protein Synthesis

Protein synthesis is a fundamental process in all living organisms. Ribosomes are the ribonucleoprotein complexes responsible for protein synthesis. Recent atomic resolution structures of the large and small ribosomal subunits provides an unique opportunity for understanding the mechanism by which ribosomes perform the complex task of protein synthesis. Ribosomes from Escherichia coli (E. coli) are used as a model system for our research. The E. coli 50S large subunit consist of a 23S rRNA molecule (2904 nucleotides), a 5S rRNA molecule (120 nucleotides) and 31 proteins. The 30S small subunit consist of a 16S rRNA molecule (1542 nucleotides) and 21 proteins. Transfer RNA (tRNA) are the substrate molecules for protein synthesis. There are three tRNA binding sites within the ribosome: Aminoacyl site (A site), Peptidyl site (P site) and Exit site (E site).

Protein synthesis consist of four major steps: (1) Initiation, (2) Elongation, (3) Termination, and (4) Ribosome recycling. During initiation, the ribosome with the help of initiation factors (IF1, IF2, and IF3) bind an mRNA and the initiator tRNA. The initiator tRNA binds to the ribosomal P site. In the elongation step, aminoacyl-tRNA corresponding to the mRNA codon in the A site binds to the ribosome. The aminoacyl-tRNA actually forms a complex with elongation factor Tu and GTP before binding to the ribosome. Once the aminoacyl-tRNA is incorporated into the ribosome, EF-Tu-GDP dissociates from the ribosome. This is a crucial step that determines the accuracy of protein synthesis. The ribosome then catalyzes peptide bond formation. After peptide bond formation, the P and A site tRNAs are translocated to the E and P sites, respectively. Translocation is catalyzed by elongation factor G. This process of iteratively binding tRNA, peptide bond formation, and translocation continues until the ribosomes encounters the stop codon in the mRNA. When the ribosome arrives at the stop codon, release factors (RF1, RF2, and RF3) bind to the ribosome and release the newly synthesized polypeptide from the ribosome. Finally, the ribosome is split into the two subunits by recycling factor (RRF, and this step also requires EF-G) and recycled for a new round of protein synthesis. How the ribosome performs each of these complex steps is not fully clear. The long-term goal of my laboratory is to elucidate the molecular mechanism of translation. We have developed powerful biochemical, biophysical and genetic tools to study specific steps in translation. Our studies will provide a better understanding of this vital cellular process.

Antibiotics: A more applied aspect of ribosome research, is the potential for discovering novel antibiotics. Bacteria cause numerous diseases such as tuberculosis, meningitis, pneumonia, leprosy, typhoid, cholera, and bubonic plague. Bacterial ribosomes are the target for inactivation by several classes of antibiotics. In fact, more than two-thirds of the currently prescribed antibiotics target bacterial ribosomes. Antibiotics such as erythromycin, spectinomycin, viomycin, thiostrepton, and the aminoglycosides (neomycin, kanamycin, etc) specifically inhibit protein synthesis. Antibiotic-resistant strains of bacteria are on the rise, causing a crisis in the management and treatment of these infections throughout the world. Understanding the mechanism of translation will provide insights for developing more effective antibiotics that target the ribosome of these drug-resistant strains of bacteria. We have developed several fluorescence-based assays to monitor defined steps of translation. These assays can be used in high-throughput screens for discovering novel inhibitors of bacterial translation.

Postdoctoral Positions Available: We are seeking talented postdoctoral fellows to study the mechanism of bacterial translation. Applicants must have a recent Ph.D. and expertise in molecular biology with publications in international peer-reviewed journals. Highly motivated applicants with experience in RNA biochemistry, enzyme kinetics, quantitative biology, and structure-function analysis are invited to apply. Please send curriculum vitae and the names of three references to Dr. Simpson Joseph, 4102 Urey Hall, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0314 USA. E-mail: sjoseph@chem.ucsd.edu

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