RRM
2006
Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
Ribosomal RNAs are the functionally important components of ribosomes. Helix 23b is located in the small-subunit rRNA and plays an important role in interactions of tRNA with the P-site and the E-site, mRNA binding, IF3 binding, and formation of the initiation complex. In addition, helix 23 is involved in intramolecular interactions with helix 24 (the 790 loop) and intermolecular interactions with ribosomal proteins and domain IV of 23S rRNA of large ribosomal subunit. The hairpin loop nucleotides of helix 23 are highly phylogenetically conserved. The nucleotide sequence of helix 23 internal loop (nucleotides 684 to 687 and 700 to 706 of Escherichia coli 16S rRNA), however, differ significantly between animals and bacteria.
The aim of this project is to identify the functionally important sequence and structural motifs within the internal loop of helix 23. This will be accomplished by integrating in vivo genetic studies with structural studies using NMR.
For genetic studies, eleven nucleotides of helix 23b internal loop were randomly mutated using PCR. The PCR products were cloned into cloning vector pwk122. A total of 3 x 107 clones were obtained to ensure the representation of mutant bank with > 99.9% confidence. The Helix 23b internal loop mutant bank was moved into expression vector pRNA228. In this vector, the chloramphenicol acetyltransferase (CAT) and the green fluorescent protein (GFP) mRNAs are translated only by plasmid derived ribosomes. CAT protein renders selectivity of mutants and GFP allows rapid and accurate determination of ribosome function by directly measuring fluorescence in whole cells. Functional mutants were selected , sequenced and assayed for GFP% function. A total of 115 unique functional mutants were isolated in the range of 13%-139% of wild type GFP function. The number of mutations per survivor ranged from two to eight nucleotides.
Keywords: Helix23b 16S rRNA, GFP function
