Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Analysis of Modification at 966 and 967 Positions in Escherichia coli 16 S ribosomal RNA

The eight-nucleotide sequence of the 970 loop of helix 31 is conserved within the 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 structure 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 functionally important nucleotides, sequence motifs and structural motifs in the 970 loop, all eight of the conserved loop nucleotides (964-971) were subjected to saturation mutagenesis, and functional mutants were selected, sequenced, and assayed. Analyses of the functional mutants revealed that single mutations at positions 966 and 967 produce ribosomes that are approximately 20%-30% more active than wild-type ribosomes. To determine the effect of these hyperactive mutations on translational fidelity, the mutant ribosomes were tested for mistranslation by starving wild-type and mutant 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 but that the m5C967U mutation has little effect on misincorporation. All three 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 three mutations at position 966 produced increased read-through of both codons but no effect on read-through was observed in the 967 mutants. Biochemical and modeling studies suggest that initiation factor 3 (IF3) interacts with the stacked residues 966:967:968. 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 effect IF3 binding to the 30 S subunit.

Tek N. Lamichhane1, 2, *
Ashesh A. Saraiya3
Christine S. Chow1
Philip R. Cunningham2

1Department of Chemistry
Wayne State University
Detroit, MI
2Department of Biological Sciences
Wayne State University
Detroit, MI
3Department of Pharmaceutical Chemistry
University of California
San Francisco, CA