Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).

[1]  R. Losick,et al.  Bacillus Subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics , 1993 .

[2]  R. Switzer,et al.  Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). , 1986, The Journal of biological chemistry.

[3]  S. Ghim,et al.  Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression , 1996, Journal of bacteriology.

[4]  C. Yanofsky,et al.  TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Min Yang,et al.  The structure of trp RNA-binding attenuation protein , 1995, Nature.

[6]  P. Nygaard.  Purine and Pyrimidine Salvage Pathways , 1993 .

[7]  R. Switzer,et al.  Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis. , 1990, European journal of biochemistry.

[8]  C. Yanofsky,et al.  Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon , 1986, Journal of bacteriology.

[9]  Y. Lu,et al.  Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  D. Mead,et al.  Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. , 1986, Protein engineering.

[11]  R. Switzer,et al.  Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. , 1991, The Journal of biological chemistry.

[12]  C. Turnbough,et al.  Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli , 1992, Journal of bacteriology.

[13]  Y. Lu,et al.  Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression , 1995, Journal of bacteriology.

[14]  C. Turnbough,et al.  Attenuation control of pyrBI operon expression in Escherichia coli K-12. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Y. Lu,et al.  Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism , 1994, Journal of bacteriology.

[16]  P. Gollnick.  Regulation of the Bacillus subtilis trp operon by an RNA‐binding protein , 1994, Molecular microbiology.

[17]  Y. Lu,et al.  Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo , 1996, Journal of bacteriology.