The structural and functional roles of post-transcriptional modifications of ribosomal RNA will be studied by site-directed mutagenesis of a cloned ribosomal RNA operon. The ribosome is responsible for protein synthesis, one of the key determinants of rapid cell growth. Inhibition of protein synthesis is the basis of action of many antibiotics. A complete understanding of the mechanisms of protein synthesis will require a knowledge of the role of rRNA and post-transcriptional modifications. With this knowledge alternative approaches to antibiotic therapeutics and design may become apparent. Three mutants will be made for each of the known sites of post- transcriptional modification in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis. Mutations will be cloned in plasmids and expressed in vivo. Their effects on RNA maturation, defined as processing of the transcript, modification of sites and incorporation into ribosomal subunits will be studied by specifically labeling transcripts from cloned genes using a modification of the maxicell technique. The rRNA will be studied from the absence of modifications or for aberrant modifications because the mutants represent altered substrates for the modifying enzymes. Modifications will be located by sequencing rRNA using DNA primers and reverse transcriptase. They will be further characterized by first, isolating short sequences of rRNA using complementary single-stranded DNA to protect regions of interest from RNase T1 digestion and second, analyzing the components of this rRNA fragment by thin layer chromatography. The ability of mutant rRNA to form functional ribosomes will be tested and effects on translational fidelity will be measured. This research will begin to define the roles of post-transcriptional modificatoins of rRNA in ribosomal structure and function.
| Jemiolo, D K; Pagel, F T; Murgola, E J (1995) UGA suppression by a mutant RNA of the large ribosomal subunit. Proc Natl Acad Sci U S A 92:12309-13 |
| Jemiolo, D K; Taurence, J S; Giese, S (1991) Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207. Nucleic Acids Res 19:4259-65 |
