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Structural and functional aspects of ribosomal protein S8-RNA interactions in Escherichia coli: Genetic, biochemical and biophysical studies
Abstract
The interaction of E. coli protein S8 with its binding sites in 16S rRNA and spc operon mRNA was studied by genetic, biochemical and biophysical approaches. Previously, about 40 S8 mutants, which are potentially defective in their RNA-binding ability, were isolated by a screening procedure based on the autogeneous regulatory role of S8. Many of mutant S8 proteins were overexpressed and purified. Under the experimental conditions I employed, S8 was expressed in cells at a level of 35% total cellular protein and could be isolated at a purity of over 98% using HPLC or a combination of ion-exchange and gel-filtration chromatography. The affinity of S8 mutants for RNA was measured by nitrocellulose filter binding assays, and the RNA-binding capacities of the mutant proteins were found to be impaired significantly. These results suggest that the RNA-binding domain of S8 consists of three phylogenetically conserved regions, composed of amino acids 4-17, 75-88, and 105-128. In addition, the affinities of mutant S8 proteins for 16S rRNA and spc mRNA decreased in parallel. This indicates that S8 utilizes the same binding domains for both RNAs. The role of the unique Cys126 residue was studied by site-directed mutagenesis, circular dichroism, and urea and thermal denaturation. These results indicate that Cys126 is unlikely to play a specific role in RNA recognition but that it is an integral part of the RNA-binding domain of S8. Many variants of the rRNA and mRNA binding sites for S8 were prepared by in vitro transcription. A 33-nucleotide "minimum" rRNA binding site, consisting of a stem-loop-stem structure, was determined by deletion analysis. The affinity of S8 for its mRNA site is lower than that for its rRNA site by one order of magnitude, although the two sites are very similar in structure. The strength of the S8-mRNA interaction appears to be modulated downward by a pair of bulged bases whose removal increases S8 affinity to a level characteristic of the rRNA site. Preliminary results on the structural analysis of S8 and the S8-rRNA complex by NMR and X-ray crystallography are presented.
Subject Area
Molecular biology|Biochemistry|Biophysics
Recommended Citation
Wu, Herren, "Structural and functional aspects of ribosomal protein S8-RNA interactions in Escherichia coli: Genetic, biochemical and biophysical studies" (1993). Doctoral Dissertations Available from Proquest. AAI9408363.
https://scholarworks.umass.edu/dissertations/AAI9408363