Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Jul 25;16(14A):6339–6352. doi: 10.1093/nar/16.14.6339

Regulation of in vitro expression of the Escherichia coli frd operon: alanine and Fnr represent positive and negative control elements.

D J Latour 1, J H Weiner 1
PMCID: PMC338299  PMID: 2456525

Abstract

The frdABCD operon of Escherichia coli encodes the anaerobically expressed terminal electron transport enzyme, fumarate reductase. Two mutually exclusive hairpin loop structures can occur in frdmRNA just downstream of the start of the frdA cistron. The mRNA sequence involved encodes a stretch of sequence rich in Ala and uses all four of the codons for this amino acid. In vitro expression of the frdABCD operon showed that as the level of plasmid DNA was increased from 150 fmol to 225 fmol, transcription of mRNA was suddenly elevated 6.5-fold, consistent with the concept of titrating out a repressor protein. Further studies showed that the concomitant 10.9-fold increase in translation of protein was heavily biased towards the proximal end of the operon, with little or no expression of FrdC or FrdD and a ratio of FrdA:FrdB of 2.6:1. Addition of Ala to the S-30 extract caused a 6.1-fold amplification of frd messenger transcription, a 17.6-fold increase in Frd protein translation, and a balancing of the subunit ratios to 1:1:1:1. The expression of the bla gene carried on the plasmid was not affected by DNA titration or the addition of Ala. When fnr DNA was added in equimolar ratio to frdDNA the amplification of fumarate reductase expression by Ala was abolished and the ratio of subunits produced showed a high degree of polarity with or without Ala.

Full text

PDF
6339

Images in this article

6344Image
on p.6344

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cole S. T., Condon C., Lemire B. D., Weiner J. H. Molecular biology, biochemistry and bioenergetics of fumarate reductase, a complex membrane-bound iron-sulfur flavoenzyme of Escherichia coli. Biochim Biophys Acta. 1985 Dec;811(4):381–403. doi: 10.1016/0304-4173(85)90008-4. [DOI] [PubMed] [Google Scholar]
  2. Cole S. T., Grundström T., Jaurin B., Robinson J. J., Weiner J. H. Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli. Eur J Biochem. 1982 Aug;126(1):211–216. doi: 10.1111/j.1432-1033.1982.tb06768.x. [DOI] [PubMed] [Google Scholar]
  3. Cole S. T. Nucleotide sequence coding for the flavoprotein subunit of the fumarate reductase of Escherichia coli. Eur J Biochem. 1982 Mar 1;122(3):479–484. doi: 10.1111/j.1432-1033.1982.tb06462.x. [DOI] [PubMed] [Google Scholar]
  4. Grundström T., Jaurin B. Overlap between ampC and frd operons on the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1111–1115. doi: 10.1073/pnas.79.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Helmann J. D., Chamberlin M. J. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6422–6424. doi: 10.1073/pnas.84.18.6422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Iuchi S., Lin E. C. The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3901–3905. doi: 10.1073/pnas.84.11.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jamieson D. J., Higgins C. F. Anaerobic and leucine-dependent expression of a peptide transport gene in Salmonella typhimurium. J Bacteriol. 1984 Oct;160(1):131–136. doi: 10.1128/jb.160.1.131-136.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jones H. M., Gunsalus R. P. Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product. J Bacteriol. 1987 Jul;169(7):3340–3349. doi: 10.1128/jb.169.7.3340-3349.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones H. M., Gunsalus R. P. Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. J Bacteriol. 1985 Dec;164(3):1100–1109. doi: 10.1128/jb.164.3.1100-1109.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klionsky D. J., Skalnik D. G., Simoni R. D. Differential translation of the genes encoding the proton-translocating ATPase of Escherichia coli. J Biol Chem. 1986 Jun 25;261(18):8096–8099. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lambden P. R., Guest J. R. Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor. J Gen Microbiol. 1976 Dec;97(2):145–160. doi: 10.1099/00221287-97-2-145. [DOI] [PubMed] [Google Scholar]
  14. Latour D. J., Weiner J. H. Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5. J Gen Microbiol. 1987 Mar;133(3):597–607. doi: 10.1099/00221287-133-3-597. [DOI] [PubMed] [Google Scholar]
  15. Lee F., Yanofsky C. Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4365–4369. doi: 10.1073/pnas.74.10.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lemire B. D., Robinson J. J., Weiner J. H. Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase. J Bacteriol. 1982 Dec;152(3):1126–1131. doi: 10.1128/jb.152.3.1126-1131.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Morse D. E., Morse A. N. Dual-control of the tryptophan operon is mediated by both tryptophanyl-tRNA synthetase and the repressor. J Mol Biol. 1976 May 15;103(2):209–226. doi: 10.1016/0022-2836(76)90310-7. [DOI] [PubMed] [Google Scholar]
  18. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  19. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ralling G., Linn T. Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region. J Bacteriol. 1987 May;169(5):2277–2280. doi: 10.1128/jb.169.5.2277-2280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  22. Ruch F. E., Kuritzkes D. R., Lin E. C. Use of lac operon fusions to isolate Escherichia coli mutants with altered expression of the fumarate reductase system in response to substrate and respiratory controls. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1365–1370. doi: 10.1016/0006-291x(79)91217-8. [DOI] [PubMed] [Google Scholar]
  23. Schmidt M. C., Chamberlin M. J. nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. J Mol Biol. 1987 Jun 20;195(4):809–818. doi: 10.1016/0022-2836(87)90486-4. [DOI] [PubMed] [Google Scholar]
  24. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Spiro S., Guest J. R. Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol. 1987 Dec;133(12):3279–3288. doi: 10.1099/00221287-133-12-3279. [DOI] [PubMed] [Google Scholar]
  26. Steitz J. A., Jakes K. How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4734–4738. doi: 10.1073/pnas.72.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  29. Unden G., Duchene A. On the role of cyclic AMP and the Fnr protein in Escherichia coli growing anaerobically. Arch Microbiol. 1987 Mar;147(2):195–200. doi: 10.1007/BF00415284. [DOI] [PubMed] [Google Scholar]
  30. Unden G., Guest J. R. Cyclic AMP and anaerobic gene expression in E. coli. FEBS Lett. 1984 May 21;170(2):321–325. doi: 10.1016/0014-5793(84)81336-8. [DOI] [PubMed] [Google Scholar]
  31. Wood D., Darlison M. G., Wilde R. J., Guest J. R. Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli. Biochem J. 1984 Sep 1;222(2):519–534. doi: 10.1042/bj2220519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yanofsky C. Attenuation in the control of expression of bacterial operons. Nature. 1981 Feb 26;289(5800):751–758. doi: 10.1038/289751a0. [DOI] [PubMed] [Google Scholar]
  33. Yanofsky C. Mutations affecting tRNATrp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon. J Mol Biol. 1977 Jul 15;113(4):663–677. doi: 10.1016/0022-2836(77)90229-7. [DOI] [PubMed] [Google Scholar]
  34. Zurawski G., Elseviers D., Stauffer G. V., Yanofsky C. Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5988–5992. doi: 10.1073/pnas.75.12.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES