One of the fundamental steps during translation is the precise and coordinated movement of the mRNA-tRNA complex within the ribosome, called translocation. The long-term goal of this research is to understand the molecular mechanism of translocation by the ribosome. Previous studies in the PI's laboratory showed that specific ribose 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the A and P site tRNAs are important for translocation. The goal of the current project is to identify 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the mRNA that are required for translocation. Additionally, the current project will directly examine the role of the universally conserved bases in 16S rRNA that interacts with the mRNA-tRNA complex in translocation. The research will use fluorescence-based pre-steady state kinetic analysis, site-directed mutagenesis of critical residues, and biochemical assays to study the mechanism of translocation. These experiments will provide information about the dynamics of ribosome structure that cannot be easily acquired by X-ray crystallography. Bacterial ribosomes are the target for inactivation by several classes of antibiotics. Antibiotics such as erythromycin, viomycin, thiostrepton and spectinomycin specifically inhibit translocation. Understanding the mechanism of translation will provide insights for developing novel antibiotics. This project also serves an important educational need by integrating research and teaching. The research will promote teaching and training in advanced biophysical methods. Several undergraduate minority students, graduate students and postdoctoral students will obtain invaluable research experience in the laboratory.
All cells require proteins for carrying out biochemical reactions essential for normal growth and division. Large complexes called ribosomes synthesize all the proteins required by the cell. Ribosomes translate the information present in messenger RNA (mRNA) by binding specific adapter molecules called transfer RNA (tRNA) that bring amino acids required for protein synthesis. This occurs in a step-wise fashion with the tRNA first binding to the A site followed by translocation to the P site before leaving the ribosome via the E site. The translocation of tRNAs through the ribosome is catalyzed by elongation factor G. The molecular mechanism of how the tRNAs are precisely translocated by the ribosome is not clear. The goal of the NSF funded project was to dissect the mechanism of mRNA-tRNA translocation by the ribosome. Specifically, we analyzed the importance of the interactions made by the mRNA with the ribosome in translocation. Our studies showed that disrupting the interactions made by the mRNA with the ribosomal A site (decoding center) accelerates translocation. In addition, we showed that universally conserved 16S rRNA nucleotides in the decoding center are important for efficient translocation. The significance of this research is that most of the antibiotics work by inhibiting bacterial protein synthesis. Understanding the mechanism of protein synthesis is critical for developing novel antibiotics to treat drug-resistant bacterial infections. The broader impact of this research is the training and development of students at all levels. The research was carried out by several undergraduate, graduate and postdoctoral students. Students received training in advanced biophysical and biochemical methods. Training students in math and science is a key aspect of the research project.
