Escherichia coli holoenzyme RNA polymerase is composed of a core enzyme (E, 1 subunit composition ␣ 2␤␤ Ј) associated with one of seven sigma ()-factors that program the complex to engage and initiate transcription at different sets of promoters (1). Thus, the levels and binding properties of alternative -subunits together with factors that modulate their ability to associate with core RNA polymerase are critical for the relative composition of the multiple holoenzymes available for transcription of the distinct promoter classes within the prokaryotic genome. The seven different -factors of E. coli fall into two groups. The larger of these comprises six factors that share notable sequence and functional similarities to the major D

The Escherichia coli proteins DksA, GreA and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation while GreA and GreB activate RNAP to cleave backtracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel binding proteins and RNAP can provide a new mode for regulating transcription.

We present evidence that Escherichia coli RNA polymerase ␤ subunit may be a transcriptional activator contact site. Stimulation of the activity of the p R promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage . We found that DnaA activates the p R promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the p R promoter was scrambled by in vitro mutagenesis, the p R promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the ␤ subunit of RNA polymerase during activation of the p R promoter. A new classification of prokaryotic transcriptional activators is proposed.Activation of transcription is a common way to regulate gene expression in both prokaryotes and eukaryotes (for reviews, see refs. 1 and 2). In bacterial cells, transcription activation at a given promoter is achieved usually by a direct contact between a transcriptional activator and RNA polymerase. The initiation of replication of plasmids derived from bacteriophage , known as plasmids (a map of the replication region present in standard plasmids is presented in Fig. 1), requires transcription at or near the origin of replication ori (for a review, see ref. 9). Transcription starting from the p R promoter and provides mRNA for production of the replication proteins O and P. Moreover, this transcription serves in the so called transcriptional activation of ori. It seems that the main regulatory role in the initiation of plasmid replication is played by the transcriptional activation of ori rather than by binding of the initiator protein O to ori (10-15). Therefore, regulation of the activity of the p R promoter is crucial for the control of plasmid DNA replication.It has been reported that E. coli DnaA protein is important for replication of bacteriophage (16) and plasmids (17,18). Subsequent studies demonstrated that transcription starting at p R and proceeding through ori is depressed in certain dnaA mutants, which led to the conclusion that this promoter is positively regulated by DnaA (19). The above mentioned conclusion (18) were strengthened by observations that wildtype plasmids cannot replicate in certain temperaturesensitive dnaA mutants even at temperatures permissive for bacterial growth (30 or 37°C) and that this defect may be suppressed by a mutation of the type in P gene. It was proposed that transcriptional activation of ori is coupled with the chaperone-mediated rearrangement of the preprimosomal complex (liberation of DnaB from P inhibition) and insertion of the preprimosome between transiently separated DNA strands (16,18). Because the product of the P gene harboring a mutation interacts significantly weaker with the host DnaB helicase than does the wild-type P protein (20), it was proposed that impaired transcript...