Probing Ribosomal Function

Hill, Walter

Abstract

The overriding purpose of this research is to elucidate the function of the ribosome. Specifically the function of exposed, single-stranded regions of ribosomal RNA will be assayed to determine how they affect the function of the ribosome. In this way the sites where tRNA, mRNA and translation factors bind can be determined. In addition, changes in the structure of the rRNA as the ribosome functions in translation can be observed. Of the ribosome functions in translation can be observed. Of particular interest are the sites where antibiotics bind, since ribosomal function or structure is markedly affected in these cases. The design of the experiments is to chemically synthesize short DNA oligomers complementary to specific regions of ribosomal RNA, hybridize these DNA probes to target sites on the ribosome, and assay the resulting affects on tRNA binding, mRNA binding translation factor binding or antibiotic binding. Relative binding will be assayed by monitoring the residual binding activity of radioactively- labeled probes or ligands using nitrocellulose filter assays. Specificity of site binding will be determined using RNase H. Using these results it should be possible to begin to understand how mRNA, tRNA, translation factors and the ribosome go about the business of synthesizing proteins.