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. 1994 Jun;176(12):3708–3722. doi: 10.1128/jb.176.12.3708-3722.1994

Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism.

R J Turner 1, Y Lu 1, R L Switzer 1
PMCID: PMC205560  PMID: 8206849

Abstract

A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (nt) untranslated leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic region; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base conserved sequence in its promoter-proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination. Deletion of pyrR from the chromosome resulted in the constitutive, elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.

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Selected References

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  1. Andersen P. S., Smith J. M., Mygind B. Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur J Biochem. 1992 Feb 15;204(1):51–56. doi: 10.1111/j.1432-1033.1992.tb16604.x. [DOI] [PubMed] [Google Scholar]
  2. Babitzke P., Yanofsky C. Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):133–137. doi: 10.1073/pnas.90.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernlohr D. A., Switzer R. L. Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo. J Bacteriol. 1983 Feb;153(2):937–949. doi: 10.1128/jb.153.2.937-949.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bond R. W., Field A. S., Switzer R. L. Nutritional regulation of degradation of aspartate transcarbamylase and of bulk protein in exponentially growing Bacillus subtilis cells. J Bacteriol. 1983 Jan;153(1):253–258. doi: 10.1128/jb.153.1.253-258.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Contente S., Dubnau D. Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. Mol Gen Genet. 1979 Jan 2;167(3):251–258. doi: 10.1007/BF00267416. [DOI] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ghim S. Y., Neuhard J. The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease. J Bacteriol. 1994 Jun;176(12):3698–3707. doi: 10.1128/jb.176.12.3698-3707.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ghim S. Y., Nielsen P., Neuhard J. Molecular characterization of pyrimidine biosynthesis genes from the thermophile Bacillus caldolyticus. Microbiology. 1994 Mar;140(Pt 3):479–491. doi: 10.1099/00221287-140-3-479. [DOI] [PubMed] [Google Scholar]
  9. Grandoni J. A., Fulmer S. B., Brizzio V., Zahler S. A., Calvo J. M. Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine. J Bacteriol. 1993 Dec;175(23):7581–7593. doi: 10.1128/jb.175.23.7581-7593.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hammer-Jespersen K., Munch-Petersen A. Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization. Mol Gen Genet. 1973 Nov 2;126(2):177–186. doi: 10.1007/BF00330992. [DOI] [PubMed] [Google Scholar]
  11. Kaptain S., Downey W. E., Tang C., Philpott C., Haile D., Orloff D. G., Harford J. B., Rouault T. A., Klausner R. D. A regulated RNA binding protein also possesses aconitase activity. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10109–10113. doi: 10.1073/pnas.88.22.10109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kern L., de Montigny J., Jund R., Lacroute F. The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene. 1990 Apr 16;88(2):149–157. doi: 10.1016/0378-1119(90)90026-n. [DOI] [PubMed] [Google Scholar]
  14. Kuroda M. I., Henner D., Yanofsky C. cis-acting sites in the transcript of the Bacillus subtilis trp operon regulate expression of the operon. J Bacteriol. 1988 Jul;170(7):3080–3088. doi: 10.1128/jb.170.7.3080-3088.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Leonhardt H., Alonso J. C. Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis. J Gen Microbiol. 1988 Mar;134(3):605–609. doi: 10.1099/00221287-134-3-605. [DOI] [PubMed] [Google Scholar]
  17. Lerner C. G., Stephenson B. T., Switzer R. L. Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster. J Bacteriol. 1987 May;169(5):2202–2206. doi: 10.1128/jb.169.5.2202-2206.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lerner C. G., Switzer R. L. Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). J Biol Chem. 1986 Aug 25;261(24):11156–11165. [PubMed] [Google Scholar]
  19. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  20. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  21. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  22. Ostrovsky de Spicer P., Maloy S. PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4295–4298. doi: 10.1073/pnas.90.9.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Otridge J., Gollnick P. MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan-dependent manner. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):128–132. doi: 10.1073/pnas.90.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paulus T. J., McGarry T. J., Shekelle P. G., Rosenzweig S., Switzer R. L. Coordinate synthesis of the enzymes of pyrimidine biosynthesis in Bacillus subtilis. J Bacteriol. 1982 Feb;149(2):775–778. doi: 10.1128/jb.149.2.775-778.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Post D. A., Hove-Jensen B., Switzer R. L. Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression. J Gen Microbiol. 1993 Feb;139(2):259–266. doi: 10.1099/00221287-139-2-259. [DOI] [PubMed] [Google Scholar]
  26. Potvin B. W., Kelleher R. J., Jr, Gooder H. Pyrimidine biosynthetic pathway of Baccillus subtilis. J Bacteriol. 1975 Aug;123(2):604–615. doi: 10.1128/jb.123.2.604-615.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Quinn C. L., Stephenson B. T., Switzer R. L. Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. J Biol Chem. 1991 May 15;266(14):9113–9127. [PubMed] [Google Scholar]
  28. Rasmussen U. B., Mygind B., Nygaard P. Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12. Biochim Biophys Acta. 1986 Apr 11;881(2):268–275. doi: 10.1016/0304-4165(86)90013-9. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shimotsu H., Kuroda M. I., Yanofsky C., Henner D. J. Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon. J Bacteriol. 1986 May;166(2):461–471. doi: 10.1128/jb.166.2.461-471.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shindler D. B., Prescott L. M. Improvements on the Prescott-Jones method for the colorimetric analysis of ureido compounds. Anal Biochem. 1979 Sep 1;97(2):421–422. doi: 10.1016/0003-2697(79)90096-4. [DOI] [PubMed] [Google Scholar]
  32. Stepanov V. M., Strongin A. Y., Izotova L. S., Abramov Z. T., Lyublinskaya L. A., Ermakova L. M., Baratova L. A., Belyanova L. P. Intracellular serine protease from Bacillus subtilis. Structural comparison with extracellular serine proteases-subtilisins. Biochem Biophys Res Commun. 1977 Jul 11;77(1):298–305. doi: 10.1016/s0006-291x(77)80196-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  35. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  36. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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