Regulation of the Escherichia coli rrnB P2 promoter

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.     

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.     

Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells

Summary Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.     

A family of expression vectors based on the rrnB P2 promoter of Escherichia coli.

We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.     

Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1.

The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.