Translation Initiation

Translation initiation is a key step for regulating the expression of many genes in the cell. In bacteria, three initiation factors (IF1, IF2, and IF3) bind to the 30S subunit and recruit the initiator tRNA and an mRNA. Then the 50S subunit binds to the 30S initiation complex triggering the release of the initiation factors from the 70S ribosome. Initiation is now complete and the ribosome enters the elongation cycle of protein synthesis.

Eventhough the 30S initiation complex has a uniform composition, the efficiency with which it recruits an mRNA is different. The primary sequence and secondary structure of the mRNAs are very divergent resulting in differences in their ability to interact productively with the 30S pre-initiation complex, which may lead to differences in the level of proteins in the cell. We are studying the interaction of initiation factors, mRNA and the initiator tRNA with the 30S subunit using fluorescence-based, rapid-kinetic methods. Our goal is to understand the molecular steps during initiation and how the initiation complex assemble on different mRNAs.

Mechanism of Translocation

One of the crucial functions performed by the ribosome is the iterative movement of the tRNA-mRNA complex, a process called translocation. The molecular mechanism of translocation is still elusive and critical interactions within the ribosome that are essential for this process is unknown. The goal of my laboratory is to elucidate the molecular basis for translocation. The different components that are involved in translocation are the mRNA, deacylated-tRNA, peptidyl-tRNA, elongation factor G (EF-G), and the ribosome. EF-G is a GTPase and the chemical energy of GTP hydrolysis is coupled to translocation of the tRNA-mRNA complex within the ribosome. Remarkably, ribosomes can translocate at a slow rate even in the absence of EF-G, indicating that the machinery required for tRNA movement is present within the ribosome. We are focusing on identifying the interactions between A and P site tRNAs and the ribosome that are important for translocation. We previously showed that the requirement for peptidyl-tRNA bound to the ribosomal A site (acceptor site) could be satisfied by a 15-nucleotide anticodon stem-loop analog (ASL4) of tRNA. We are using synthetic variants of this A site tRNA analog to elucidate the identity and contribution of specific functional groups within A site tRNA that are essential for translocation. A related goal is to identify functional groups within P site tRNA that are required for translocation. Our studies show that tRNA having a specific break in the backbone chain are translocated when bound to the ribosomal P site. We are using this innovative approach to systematically identify functional groups in the acceptor arm of P site tRNA that are essential for translocation by site-specific incorporation of modified nucleotides within the shorter tRNA fragment. These studies will provide a frame work for understanding the importance of tRNA-ribosome interactions in translocation.

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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