1887

Abstract

We have previously cloned and sequenced the gene, encoding inorganic pyrophosphatase (PPase), of K12 [Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) 170, 5901–5907]. In this work mutations were constructed in the 5′ flanking region of and the effect on expression was determined. The minimum length of the fully active 5′ flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide –37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide –50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the –50 to +20 region in genes. When the –35 sequence of , AAGACA, was mutated to AAAACA, expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the –10 sequence, TATAAT, to TTTAAT or TATAAA, the gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both mRNA and PPase decreased drastically. The implications of these results are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-137-11-2517
1991-11-01
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/137/11/mic-137-11-2517.html?itemId=/content/journal/micro/10.1099/00221287-137-11-2517&mimeType=html&fmt=ahah

References

  1. Airas R. K., Cramer F. 1986; Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa . European Journal of Biochemistry 160:291–296
    [Google Scholar]
  2. Belasco J. G., Higgins C. F. 1988; Mechanism of mRNA decay in bacteria: a perspective. Gene 72:15–23
    [Google Scholar]
  3. Bourguignon-Van Horen F., Brotcorn A., Caillet-Fauquet P., Diver W. P., Dohet C, Doupleday O. P., Lecomte P., Maenhaut-Michel G., Radman M. 1982; Conservation and diversification of genes by mismatch correction and SOS induction. Biochimie 64:559–564
    [Google Scholar]
  4. Brunner M., Bujard H. 1987; Promoter recognition and promoter strength in the Escherichia coli system. EMBO Journal 6:3139–3144
    [Google Scholar]
  5. Chen J., Brevet A., Fromant M., Leveque F., Schmitter J.-M., Blanquet S., Plateau P. 1990; Pyrophosphatase is essential to the growth of Escherichia coli . Journal of Bacteriology 111:5686–5689
    [Google Scholar]
  6. Cooperman B. S. 1982; The mechanism of action of yeast inorganic pyrophosphatase. Methods in Enzymology 87:526–548
    [Google Scholar]
  7. Dignam J. D., Deutscher M. P. 1979; Aminoacyl-tRNA synthetase stimulatory factors and inorganic pyrophosphatase. Biochemistry 18:3165–3170
    [Google Scholar]
  8. Gaal T., Barkei J., Dickson R. R., DeBoer H. A., DeHaseth P. L., Alavi H., Course R. L. 1989; Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants. Journal of Bacteriology 171:4852–4861
    [Google Scholar]
  9. Gonzales M. A., Cooperman B. S. 1986; Glutamic acid 149 is important for the enzymatic activity of yeast inorganic pyrophosphatase. Biochemistry 25:7179–7185
    [Google Scholar]
  10. Gutterson N. I., Koshland D. E. Jr 1983; Replacement and amplification of bacterial genes with sequences altered in vitro . Proceedings of the National Academy of Sciences of the United States of America 804894–4898
    [Google Scholar]
  11. Harley C. B., Reynolds R. P. 1987; Analysis of E. coli promoter sequences. Nucleic Acids Research 15:2343–2361
    [Google Scholar]
  12. Heinonen J., Kukko E. 1977; Partial inhibition of DNA synthesis gives rise to increase in the level of inorganic pyrophosphatase in the growing cells of Escherichia coli . Chemico and Biological Interactions 17:113–116
    [Google Scholar]
  13. Heinonen J., Lahti R. 1981; A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Analytical Biochemistry 113:313–317
    [Google Scholar]
  14. Henikoff S. 1984; Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359
    [Google Scholar]
  15. Herbomel P., Ninio J. 1980; Fidelity of a polymerisation reaction in relation to proximity to equilibrium. Comptes Rendus de l’Académie des Sciences D291:881–884
    [Google Scholar]
  16. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. 1984; Protein-nucleic acid interactions in transcription: a molecular analysis. Annual Review of Biochemistry 53:389–446
    [Google Scholar]
  17. Igo M. M., Losick R. 1986; Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis . Journal of Molecular Biology 191:615–624
    [Google Scholar]
  18. Josse J., Wong S. C. K. 1971; Inorganic pyrophosphatase of Escherichia coli . The Enzymes 4:499–527 Boyer P. D. New York: Academic Press;
    [Google Scholar]
  19. Kent R. B., Guterman S. K. 1982; Pyrophosphate inhibition of ATPase : a mechanism of coupling to RNA polymerase activity. Proceedings of the National Academy of Sciences of the United States of America 793992–3996
    [Google Scholar]
  20. Klemme J. H. 1976; Regulation of intracellular pyrophosphatase-activity and conservation of the phosphoanhydride-energy of inorganic pyrophosphate in microbial metabolism. Zeitschrift für Naturforschung C31:544–550
    [Google Scholar]
  21. Knappe J., Sawers G. 1990; A radical-chemical route to acetyl-CoA : the anaerobically induced pyruvate formate-lyase system of Escherichia coli . FEMS Microbiology Reviews 75:383–398
    [Google Scholar]
  22. Kornberg A. 1962; On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. Horizons in Biochemistry251–264 Kasha H., Pullman P. New York: Academic Press;
    [Google Scholar]
  23. Kukko-Kalske E., Heinonen J. 1985; Inorganic pyrophosphate and inorganic pyrophosphatase in Escherichia coli . International Journal of Biochemistry 17:575–580
    [Google Scholar]
  24. Kukko-Kalske E., Lintunen M., Karjalainen M., Lahti R., Heinonen J. 1989; Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells. Journal of Bacteriology 171:4498–4500
    [Google Scholar]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:376–382
    [Google Scholar]
  26. Lahti R. 1983; Microbial inorganic pyrophosphatases. Microbiological Reviews 47:169–179
    [Google Scholar]
  27. Lahti R., Jokinen M. 1985; Kinetic model for the action of the inorganic pyrophosphatase from Streptococcus faecalis . Biochemistry 24:3526–3530
    [Google Scholar]
  28. Lahti R., Pitkäranta T., Valve E., Ilta I., Kukko-Kalske E., Heinonen J. 1988; Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12. Journal of Bacteriology 170:5901–5907
    [Google Scholar]
  29. Lahti R., Kolakowski L. F., Heinonen J., Vihinen M., Pohjanoksa K., Cooperman B. 1990a; Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases. Biochimica et Biophysica Acta 1038:338–345
    [Google Scholar]
  30. Lahti R., Pohjanoksa K., Pitkäranta T., Heikinheimo P., Salminen T., Meyer P., Heinonen J. 1990b; A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid 98 and lysine 104 are important for structural integrity, whereas aspartic acids 97 and 102 are essential for catalytic activity. Biochemistry 29:5761–5766
    [Google Scholar]
  31. Lahti R., Salminen T., Latonen S., Heikinheimo P., Pohjanoksa K., Heinonen J. 1991; Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyrl41 are important for the structural integrity. European Journal of Biochemistry 198:293–297
    [Google Scholar]
  32. Lecomte P., Doupleday O. P., Radman M. 1986; Evidence for an intermediate in DNA synthesis involving pyrophosphate exchange. A possible role in fidelity. Journal of Molecular Biology 189:643–652
    [Google Scholar]
  33. Magasanik B., Neidthardt F. C. 1987; Regulation of carbon and nitrogen utilization. Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology1318–1325 Ingraham J. L., Low K. B., Magasanik B., Schaechter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  34. Makino K., Shinagawa H., Amemura M., Nakata A. 1986; Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. Journal of Molecular Biology 190:37–44
    [Google Scholar]
  35. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Messing J. 1983; New M13 vectors for cloning. Methods in Enzymology 101:20–78
    [Google Scholar]
  37. Norrander J., Kempe T., Messing J. 1983; Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106
    [Google Scholar]
  38. Peller L. 1976; On the free-energy changes in the synthesis and degradation of nucleic acids. Biochemistry 15:141–146
    [Google Scholar]
  39. Reznikoff W. S., McClure W. R. 1986; E. coli promoters. Maximizing Gene Expression1–33 Reznikoff W., Gold L. London: Butterworths;
    [Google Scholar]
  40. Rodriquez R. L., Tait R. C. 1983 Recombinant DNA Techniques: An Introduction149 Addison-Wesley;
    [Google Scholar]
  41. Samejima T., Tamagawa Y., Kondo Y., Hachimori A., Kaji H., Takeda A., Shiroya Y. 1988; Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli . Journal of Biochemistry 103:766–772
    [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 745463–5467
    [Google Scholar]
  43. Shine J., Dalgarno L. 1974; The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA : complementarity to nonsense triplets and ribosome binding sites. Proceedings of the National Academy of Sciences of the United States of America 711342–1346
    [Google Scholar]
  44. STORMO G. D. 1986; Translation initiation. Maximizing Gene Expression195–224 Reznikoff W., Gold L. London: Butterworths;
    [Google Scholar]
  45. Syvänen M., Hopkins J. D., Clements M. 1982; A new class of mutants in DNA polymerase I that affects gene transposition. Journal of Molecular Biology 158:203–212
    [Google Scholar]
  46. Terzyan S. S., Voronova A. A., Smirnova E. A., Kuranova I. P., Nekrasov Yu. V., Arutyunyan E. G., Vainstein B. K., Hohne W., Hansen G. 1984; Spatial structure of yeast inorganic pyrophosphatase at a resolution of 3 Å. Bioorganicheskaya Khimiya 10:1469–1482
    [Google Scholar]
  47. Welsh K. M., Jacobyansky A., Springs B., Cooperman B. S. 1983; Catalytic specificity of yeast inorganic pyrophosphatase for Mg2+ as cofactor. An analysis of divalent metal ion and solvent isotope effects on enzyme function. Biochemistry 22:2243–2248
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-137-11-2517
Loading
/content/journal/micro/10.1099/00221287-137-11-2517
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error