UBC Theses and Dissertations
Essential genes in the rif region of the Escherichia coli chromosome Downing, Willa Lee
Regulation of the contiguous secE-nusG and rplKATL-rpoBC operons, found in the rif region at 90 minutes on the Escherichia coli chromosome, was examined. SecE protein is important in protein export. NusG protein is involved in transcription antitermination. The rplKATL-rpoBC gene cluster encodes, respectively, the four 50S subunit ribosomal proteins L11, L1, L10 and L12, and the β and β' subunits of RNA polymerase. The nucleotide sequences of the secE and nusG genes were determined and their transcripts were analyzed by primer extension and SI nuclease mapping. The two genes are cotranscribed, with transcripts initiated at the P[sub EG] promoter and terminated at the Rho-independent terminator overlapping the P[sub L11] promoter. The majority of transcripts are processed in the 5' untranslated leader region by RNaseIII and possibly by a second unidentified nuclease. Transcripts from the rplKATL-rpoBC gene cluster were quantitated by filter hybridization and their ends mapped by SI nuclease protection. The most abundant transcript was the 2600 nucleotide tetracistronic L11-L1-L10-L12 mRNA initiated at the P[sub L11] promoter and terminated at the attenuator in the L12-β intergenic space. Less abundant 1300 nucleotide L11-L1and L10-L12 bicistronic transcripts were also observed. Two 5' ends for the L10-L12 bicistronic mRNA were located, one at the P[sub L10] promoter and the other 150 nucleotides downstream of P[sub L10’], in a region where no promoter activity has been detected. About 80% of the transcripts were terminated at the attenuator; transcripts reading through the attenuator were partially processed by RNaseDI. No other major 5' ends were observed in the L12- β intergenic region. During restriction of RNA polymerase activity, transcriptional disruption of rplKATL and rpoBC results mainly from modulation in the frequency of initiation at P[sub L11] and P[sub L10] promoters, and termination and antitermination at the attenuator. The rplTL transcript leader region is thought to mediate regulation of L10 and L12 synthesis by folding into a translationally closed or open secondary structure (T. Christensen, M. Johnsen, N.P. Fiil and J.D. Friesen (1984) EMBO J. 3, 1609-1612). Point mutants in the leader mRNA were created by site-directed mutagenesis and analyzed in an in vitro translation assay. Preliminary results suggest that alternative secondary or higher order RNA interactions may be involved.
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