Identification of factors involved in processing of mRNA in a fimbrial operon of Escherichia coli

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Identification of factors involved in processing of mRNA in a fimbrial operon of Escherichia coli

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Title: Identification of factors involved in processing of mRNA in a fimbrial operon of Escherichia coli
Author: Koo, Jovanka Tepavcevic
Abstract: Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for coordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease and involves translation of daaP which spans the processing site. It was previously determined that the amino acid sequence of DaaP downstream of the processing site is required for mRNA processing. Here, we show by alanine scanning mutagenesis of the final ten codons of daaP that the important amino acids for processing are G49, P50 and P51. When these codons were changed to an alanine processing of the mRNA was abolished, while synonymous substitutions in these codons had no effect on processing. This suggested the hypothesis that the DaaP peptide interacts with a ribosome or an associated component and facilitates cleavage of the daa mRNA in the ribosome.Here we also present results of a genetic strategy used to identify factors involved in daa mRNA processing. We utilized a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. Hfr crosses and P1 transductions were employed to locate the relevant mutation, and the mutation responsible for the processing defect was mapped to the 32 min region on E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore in trans the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore processing in the mutant.We have also identified ribosome associated factor SsrA as being involved in processing of daa mRNA. Mutant deficient in SsrA results in defective daa mRNA processing. The processing defect in this strains could be complemented by providing a wild type copy of ssrA on a plasmid. The cleavage defect in SsrA deficient strain is augmented by a mutation in hrpA, providing further evidence that SsrA is required for efficient processing of daa mRNA.
Description: Thesis (Ph. D.)--University of Washington, 2004
URI: http://hdl.handle.net/1773/11506

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