Poster abstracts

Poster number 35 submitted by Tek Lamichhane

Role of modified nucleotides in helix 31 of Escherichia coli 16 S ribosomal RNA

Tek N. Lamichhane (Department of Chemistry/Biological Sciences, Wayne State University), Ashesh A. Saraiya (Department of Pharmaceutical Chemistry, University of California, San Francisco), Christine S. Chow (Department of Chemistry), Philip R. Cunningham (Department of Biological Sciences, Wayne State University)

Abstract:
The 970 loop of helix 31 is highly conserved within Bacteria, Archaea and Eukarya, but only A964, A969, and C970 are conserved among all three domains. The 970 loop also contains two of the eleven modified nucleosides in E. coli 16 S rRNA, m2G966 and m5C967. Biochemical and structural studies have placed this loop near the P-site and have shown that it is involved in the decoding process and in binding the antibiotic tetracycline. To identify the function of modified nucleotides in this loop, mutations were constructed at m2G966 and m5C967 and tested for ribosome function in vivo using the specialized genetic system developed in the Cunningham lab. Analyses of the mutants revealed that single mutations at positions 966 and 967 produce ribosomes that are approximately 20%-30% more active than wild-type ribosomes. Biochemical and modeling studies suggest that initiation factor 3 (IF3) interacts with the stacked residues 966:967:968 in H31. Over-expression of IF3 specifically restores wild-type levels of protein synthesis to the 966 and 967 hyperactive mutants. These data suggest that m2G966 and m5C967 may affect IF3 binding to the 30 S subunit. To determine the effect of these hyperactive mutations on translational fidelity, the mutant ribosomes were tested for mistranslation by starving wild-type and mutant plasmid containing cells for asparagine and measuring amino acid misincorporation in by isoelectric focusing. These data show that the m2G966U mutation produces ribosomes that are significantly more error prone than wild-type ribosomes. All 3 mutations at positions 966 and 967 were also tested for read-through of the UAG and UGA stop codons in vivo using luciferase premature termination constructs. All 966 mutants produced increased read-through of both codons but less effect on read-through was observed with the 967 mutants. From structural studies, it is reported that m2G966 and C1400 are involved in forming a binding cavity for P-site tRNA and the distance between these two nucleotides is decreased upon tRNA binding. To determine if a functional interaction exists between m2G966 and C1400, a series of double mutants at these positions were constructed and analyzed. No significant difference in function between the double and single mutants were found. This results suggest that a functional interaction between m2G966 and C1400 does not exist.

Keywords: Ribosomal RNA, Modifying enzymes, Aminoacid starvation