An Escherichia coli gene in search of a function
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Summary
The rrB gene of Escherichia coli is preceded by an open reading frame, which is contranscribed with rrnB both in vivo and in vitro. It has earlier been shown that a 289 amino acid protein corresponding to this gene is actually synthesized in E. coli. In this paper we show that: (1.) The transcription of this gene diminishes the stringent response of the P1 promoter of the linked rrB gene, but this is a cis effect and is not mediated by the protein product of the gene. (2.) The functional integrity of this gene seems to be essential, because efforts to replace it by a plasmidcoded copy mutagenized by Tn5 completely failed. (3.) The protein product of this gene was strongly overproduced by a recombinant plasmid, exploiting the principle of “translational coupling”. This overproduction did not change the phenotype of the host cell significantly. The protein was purified to apparent electrophoretic homogenity.
Key words
rrnB gene ORFI protein Tn5 mutagenesis Stringent response Translational couplingPreview
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References
- Boros I, Csordás-Tóth É, Kiss A, Török I, Udvardy A, Udvardy K, Venetianer P (1983a) Identification of two new promoters probably involved in the transcription of a ribosomal RNA gene of Escherichia coli. Biochim Biophys Acta 739:173–180Google Scholar
- Boros I, Kiss A, Sain B, Somlyai G, Venetianer P (1983b) Cloning of the promoters of an Escherichia coli rRNA gene. New experimental system to study the regulation of rRNA transcription. Gene 22:191–201Google Scholar
- Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:41–459, 472Google Scholar
- Brosius J, Dull TJ, Sleter DD, Noller HF (1981) Gene organization and primary structure of a ribosomal RNA operon from E. coli. J Mol Biol 148:107–127Google Scholar
- de Bruijn FJ, Lupski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids — a review. Gene 27:131–149Google Scholar
- Erdei S, Boros I, Szabó G, Venetianer P (1983) A novel type of bacterial tanscription unit specifying mRNA, rRNA and tRNA. Mol Gen Genet 191:162–164Google Scholar
- Fiil N, Friesen JD (1968) Isolation of relaxed mutants of Escherichia coli. J Mol Biol 95:729–731Google Scholar
- Gourse RL, Stark MJR, Dahlberg AE (1983) Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo. Cell 32:1347–1357Google Scholar
- Kajitani M, Ishihama A (1984) Promoter selectivy of Escherichia coli RNA polymerase. Differential stringent control of multiple promoters from ribosomal RNA and protein operons. J Biol Chem 259:1951–1959Google Scholar
- Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
- Lukacsovich T, Boros I, Venetianer P (1987) New regulatory features of the promoters of an E. coli rRNA gene. J Bacteriol 169:272–277Google Scholar
- Maniatis T, Jeffrey A, Van de Sande H (1975) Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14:3787–3794Google Scholar
- Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New YorkGoogle Scholar
- Oppenheim DS, Yanofsky C (1980) Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics 95:785–795Google Scholar
- Sarmientos P, Sylvester JE, Contente S, Cashel M (1983) Differential stringent control of the tandem E. coli ribosomal RNA promoters from the rrnA operon expressed in vivo in multicopy plasmids. Cell 32:1337–1346Google Scholar
- Schümperli D, McKenney K, Sobieski DA, Rosenberg M (1982) Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon. Cell 30:865–871Google Scholar
- Szabó G, Venetianer P (1985) Translational coupling at the intercistronic boundary of an artificially constructed operon in Escherichia coli. Acta Biochim Biophys Acad Sci Hung 20:223–230Google Scholar
- Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119Google Scholar