The number of G residues in the Bacillus subtilis pyrG initially transcribed region governs reiterative transcription-mediated regulation.

Derepression of pyrG expression in Bacillus subtilis involves CTP-sensitive reiterative transcription, which introduces up to 11 extra G residues at the 5' ends of pyrG transcripts. Insertion of three or more additional Gs at the 5' end of the pyrG initially transcribed region abolished reiterative transcription and caused constitutive expression.

Mutations affecting transcription pausing in the Bacillus subtilis pyr operon.

Pausing during transcription of the Bacillus subtilis pyr operon was proposed to play a role in its regulation by attenuation. Substitution mutations in the B. subtilis pyr DNA specifying the 3'-terminal nucleotides of the previously identified transcription pause sites substantially reduced pausing at these sites in vitro. This result confirms the general utility of this mutagenic strategy for studying transcriptional pausing. Pyrimidine-mediated repression in vivo of pyr-lacZ fusions containing some of these substitution mutations was substantially lower than those observed with the wild-type pyr-lacZ fusions. However, these defects in regulation were correlated with alterations in the stability of the terminator stem-loop specified by the attenuator, rather than with their effects on transcriptional pausing in vitro.

CTP limitation increases expression of CTP synthase in Lactococcus lactis.

CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein syntheses are inhibited. Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes. In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing an antiterminator to form and transcription to proceed. This model therefore does not include any trans-acting protein for sensing the CTP concentration as previously proposed for Bacillus subtilis.