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) opero n from Bacillus subtilis was reconstituted with an in vitro system tha t consisted of pyr DNA templates, B. subtilis RNA polymerase, four rib onucleoside triphosphates, and the purified B. subtilis PyrR regulator y protein. The templates used each specified one of the three known at tenuation regions of the pyr operon. Runoff (read-though) and terminat ed transcripts of the predicted lengths were the only major products s ynthesized. 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 combin ation of PyR plus UMP. The concentration of UMP required for half-maxi mal effect was 2.5 mu M. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UD P was only effective at 100 times the concentration of UMP. Other pyri midine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferas e activity of PyrR, antagonized UMP-dependent transcriptional terminat ion, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr a ttenuation regions. The results strongly support the model for transcr iptional 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).