Fine structure of E. coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove

The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding. On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.     

Streptomyces aureofaciens sporulation-specific sigma factor sigma(rpoZ) directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds.

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, sigma(RpoZ), of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P(ren24), P(ren57), and P(ren71), contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P(ren71) was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P(ren71) promoter is recognized by an RNA polymerase holoenzyme containing sigma(RpoZ) in S. aureofaciens. Sequence analysis of the region directed by P(ren71) revealed a gene, ren71, encoding a protein of 358 amino acids with an Mr 37,770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.     

Enhancement of RNA synthesis by promoter duplication in tombusviruses

Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants, Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses.