Regulation of carAB operon expression in Escherichia coli K-12 by reiterative transcription and characterization of the reaction.

摘要

In Escherichia coli, several operons including pyrBI, codBA, UPP, and gal have been shown to be regulated by UTP-sensitive reiterative transcription. In the regulatory mechanism, high levels of intracellular UTP concentration induce reiterative transcription during initiation which inhibits productive initiation. In contrast, low levels of UTP inhibit reiterative transcription and thus facilitate productive initiation. In this study, we examined the regulatory mechanism of carAB operon, which encodes the two subunits of carbamylphosphate synthetase needed for pyrimidine nucleotide and arginine biosynthesis. Expression of the carAB operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1. In this study, the molecular mechanism for the pyrimidine-mediated regulation of carAB expression was investigated. We show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription. The sequence of the initially transcribed region of promoter P1 is 5' -GTTTGC (non-template strand). High levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUUn (where n = 1 to >30). These transcripts are not extended downstream to include structural gene sequences. In contrast, lower levels of UTP enhance normal transcript elongation and the synthesis of translatable carAB transcripts. The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor.;The promoter elements, which determine the extent of reiterative transcription, were examined. Our results show that the nucleotide sequences at transcription initiation sites which allow a stronger base-paring between the 5' -end of the RNA and the DNA template inhibit reiterative transcription and reiterative transcription-mediated gene regulation. We also found that the spacing between the -10 region and transcription initiation site strongly affect reiterative transcription and gene regulation. These results indicate that the relative positioning of the promoter binding site and the active site of the RNA is an important factor in reiterative transcription and reiterative transcription-mediated gene regulation.;We also examined the effect of GreA on reiterative transcription and reiterative transcription-mediated gene regulation of pyrBI, codBA , and carAB promoter P1. Our results show that GreA can induce cleavage of reiterative transcripts by RNA polymerase in vitro. When examined in vivo, we found that GreA, but not GreB, specifically enhances gene expression from pyrBI, codBA, and carAB promoter P1 only under conditions in which reiterative transcription is induced. These results indicate that GreA is involved in the regulation of reiterative transcription.