Abstract
The pattern of transcription of the rplKAJLrpoBC gene cluster of Escherichia coli appears to be complex. At least four different promoters and a transcriptional attenuator have been identified. To compare the relative effect of each of the putative promoters and the attenuator on transcription of these genes, we fused these regulatory sites to lacZ. These transcriptional fusions were constructed on lambda transducing phages so a single copy of each could be stably integrated into the chromosome. The level of beta-galactosidase in a lysogen of each phage reflects the activity of the transcriptional regulatory site. We find that the promoters preceding rplK (rplKp) and rplJ (rplJp) are indeed the major promoters of this gene cluster. The minor promoter before rplL (rplLp) is much weaker and contributes little to the transcription of the downstream genes. Under these conditions, we find no evidence of a promoter (rpoBp) in the rplL-rpoB intercistronic region. The attenuator (atn) terminates ca. 70% of the transcripts initiated at the promoters preceding it. Although we cannot rule out that some transcripts from rplKp may read through into rplJLrpoBC, we find that rplJp alone is sufficient for high-level expression of these genes.
Full text
PDFSelected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adhya S., Gottesman M. Promoter occlusion: transcription through a promoter may inhibit its activity. Cell. 1982 Jul;29(3):939–944. doi: 10.1016/0092-8674(82)90456-1. [DOI] [PubMed] [Google Scholar]
- An G., Friesen J. D. Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant. J Bacteriol. 1980 Dec;144(3):904–916. doi: 10.1128/jb.144.3.904-916.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barnes W. M., Siegel R. B., Reznikoff W. S. The construction of lambda transducing phages containing deletions defining regulatory elements of the lac and trp operons in E. coli. Mol Gen Genet. 1974 Mar 27;129(3):201–215. doi: 10.1007/BF00267913. [DOI] [PubMed] [Google Scholar]
- Barry G., Squires C. L., Squires C. Control features within the rplJL-rpoBC transcription unit of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4922–4926. doi: 10.1073/pnas.76.10.4922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barry G., Squires C., Squires C. L. Attenuation and processing of RNA from the rplJL--rpoBC transcription unit of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3331–3335. doi: 10.1073/pnas.77.6.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borck K., Beggs J. D., Brammar W. J., Hopkins A. S., Murray N. E. The construction in vitro of transducing derivatives of phage lambda. Mol Gen Genet. 1976 Jul 23;146(2):199–207. doi: 10.1007/BF00268089. [DOI] [PubMed] [Google Scholar]
- Brot N., Caldwell P., Weissbach H. Autogenous control of Escherichia coli ribosomal protein L10 synthesis in vitro. Proc Natl Acad Sci U S A. 1980 May;77(5):2592–2595. doi: 10.1073/pnas.77.5.2592. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brückner R., Matzura H. In vivo synthesis of a polycistronic messenger RNA for the ribosomal proteins L11, L1, L10 and L7/12 in Escherichia coli. Mol Gen Genet. 1981;183(2):277–282. doi: 10.1007/BF00270629. [DOI] [PubMed] [Google Scholar]
- Burton Z. F., Gross C. A., Watanabe K. K., Burgess R. R. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12. Cell. 1983 Feb;32(2):335–349. doi: 10.1016/0092-8674(83)90453-1. [DOI] [PubMed] [Google Scholar]
- Crawford I. P., Nichols B. P., Yanofsky C. Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium. J Mol Biol. 1980 Oct 5;142(4):489–502. doi: 10.1016/0022-2836(80)90259-4. [DOI] [PubMed] [Google Scholar]
- Dennis P. P. Transcription patterns of adjacent segments on the chromosome of Escherichia coli containing genes coding for four 50S ribosomal proteins and the beta and beta' subunits of RNA polymerase. J Mol Biol. 1977 Oct 5;115(4):603–625. doi: 10.1016/0022-2836(77)90105-x. [DOI] [PubMed] [Google Scholar]
- Fiil N. P., Bendiak D., Collins J., Friesen J. D. Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids. Mol Gen Genet. 1979 May 23;173(1):39–50. doi: 10.1007/BF00267689. [DOI] [PubMed] [Google Scholar]
- Friesen J. D., An G., Fiil N. The lethal effect of a plasmid resulting from transcriptional readthrough of rplJ from the rplKA operon in Escherichia coli. Mol Gen Genet. 1983;189(2):275–281. doi: 10.1007/BF00337817. [DOI] [PubMed] [Google Scholar]
- Goldberg G., Zarucki-Schulz T., Caldwell P., Weissbach H., Brot N. Regulation of the in vitro synthesis of E. coli ribosomal protein L12. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1453–1461. doi: 10.1016/0006-291x(79)91229-4. [DOI] [PubMed] [Google Scholar]
- Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
- Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
- Holowachuk E. W., Friesen J. D., Fiil N. P. Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2124–2128. doi: 10.1073/pnas.77.4.2124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howe K. M., Newman A. J., Garner I., Wallis A., Hayward R. S. Effect of rifampicin on expression of lacZ fused to promoters or terminators of the E.coli rpoBC operon. Nucleic Acids Res. 1982 Nov 25;10(22):7425–7438. doi: 10.1093/nar/10.22.7425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hui I., Maltman K., Little R., Hastrup S., Johnsen M., Fiil N., Dennis P. Insertions of transposon Tn5 into ribosomal protein PNA polymerase operons. J Bacteriol. 1982 Dec;152(3):1022–1032. doi: 10.1128/jb.152.3.1022-1032.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jaskunas S. R., Burgess R. R., Nomura M. Identification of a gene for the alpha-subunit of RNA polymerase at the str-spc region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1975 Dec;72(12):5036–5040. doi: 10.1073/pnas.72.12.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschbaum J. B., Konrad E. B. Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J Bacteriol. 1973 Nov;116(2):517–526. doi: 10.1128/jb.116.2.517-526.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindahl L., Zengel J. M. Expression of ribosomal genes in bacteria. Adv Genet. 1982;21:53–121. doi: 10.1016/s0065-2660(08)60297-7. [DOI] [PubMed] [Google Scholar]
- Linn T., Goman M., Scaife J. G. Studies on the control of the genes for transcription and translation in Escherichia coli K12 I. tufB and rplA, K have separate promoters. J Mol Biol. 1979 Jun 5;130(4):405–420. doi: 10.1016/0022-2836(79)90431-5. [DOI] [PubMed] [Google Scholar]
- Linn T., Scaife J. Identification of a single promoter in E. coli for rplJ, rplL and rpoBC. Nature. 1978 Nov 2;276(5683):33–37. doi: 10.1038/276033a0. [DOI] [PubMed] [Google Scholar]
- Ma J. C., Newman A. J., Hayward R. S. Internal promoters of the rpoBC operon of Escherichia coli. Mol Gen Genet. 1981;184(3):548–550. doi: 10.1007/BF00352538. [DOI] [PubMed] [Google Scholar]
- Mitchell D. H., Reznikoff W. S., Beckwith J. R. Genetic fusions defining trp and lac operon regulatory elements. J Mol Biol. 1975 Apr 15;93(3):331–350. doi: 10.1016/0022-2836(75)90281-8. [DOI] [PubMed] [Google Scholar]
- Newman A. J., Linn T. G., Hayward R. S. Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli. Mol Gen Genet. 1979 Jan 31;169(2):195–204. doi: 10.1007/BF00271671. [DOI] [PubMed] [Google Scholar]
- Ovchinnikov Y. A., Monastyrskaya G. S., Gubanov V. V., Guryev S. O., Chertov OYu, Modyanov N. N., Grinkevich V. A., Makarova I. A., Marchenko T. V., Polovnikova I. N. The primary structure of Escherichia coli RNA polymerase. Nucleotide sequence of the rpoB gene and amino-acid sequence of the beta-subunit. Eur J Biochem. 1981 Jun 1;116(3):621–629. doi: 10.1111/j.1432-1033.1981.tb05381.x. [DOI] [PubMed] [Google Scholar]
- Ovchinnikov Y. A., Monastyrskaya G. S., Guriev S. O., Kalinina N. F., Sverdlov E. D., Gragerov A. I., Bass I. A., Kiver I. F., Moiseyeva E. P., Igumnov V. N. RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance. Mol Gen Genet. 1983;190(2):344–348. doi: 10.1007/BF00330662. [DOI] [PubMed] [Google Scholar]
- Post L. E., Strycharz G. D., Nomura M., Lewis H., Dennis P. P. Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1697–1701. doi: 10.1073/pnas.76.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith H. O. Recovery of DNA from gels. Methods Enzymol. 1980;65(1):371–380. doi: 10.1016/s0076-6879(80)65048-4. [DOI] [PubMed] [Google Scholar]
- Taylor W. E., Burgess R. R. Escherichia coli RNA polymerase binding and initiation of transcription on fragments of lambda rifd 18 DNA containing promoters for lambda genes and for rrnB, tufB, rplC,A, rplJ,L, and rpoB,C genes. Gene. 1979 Aug;6(4):331–365. doi: 10.1016/0378-1119(79)90073-8. [DOI] [PubMed] [Google Scholar]
- Wilson G. G., Murray N. E. Molecular cloning of the DNA ligase gene from bacteriophage T4. I. Characterisation of the recombinants. J Mol Biol. 1979 Aug 15;132(3):471–491. doi: 10.1016/0022-2836(79)90270-5. [DOI] [PubMed] [Google Scholar]
- Windass J. D., Brammar W. J. Aberrant immunity behaviour of hybrid lambda imm21 phages containing the DNA of ColE1-type plasmids. Mol Gen Genet. 1979;172(3):329–337. doi: 10.1007/BF00271733. [DOI] [PubMed] [Google Scholar]
- Yamamoto M., Nomura M. Contranscription of genes for RNA polymerase subunits beta and beta' with genes for ribosomal proteins in Escherichia coli. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3891–3895. doi: 10.1073/pnas.75.8.3891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto M., Nomura M. Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome. J Bacteriol. 1979 Jan;137(1):584–594. doi: 10.1128/jb.137.1.584-594.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanofsky C., Horn V., Bonner M., Stasiowski S. Polarity and enzyme functions in mutants of the first three genes of the tryptophan operon of Escherichia coli. Genetics. 1971 Dec;69(4):409–433. doi: 10.1093/genetics/69.4.409. [DOI] [PMC free article] [PubMed] [Google Scholar]