The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression

Background: Among the seven different sigma factors in E. coli s 70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting s 70-dependent transcription at the onset of stationary growth. Although binding of Rsd to s 70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings: We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with s 70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with s 38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and s 38-ors 70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of s 70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned ou

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

Oligomerization Mediated by a Helix-Loop-Helix-Like Domain of Baculovirus IE1 Is Required for Early Promoter Transactivation

IE1 is a principal transcriptional regulator of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transactivation by IE1 is stimulated when early viral promoters are cis linked to homologous-region (hr) enhancer sequences of AcMNPV. This transcriptional enhancement is correlated with the binding of IE1 as a dimer to the 28-bp palindromic repeats comprising the hr enhancer. To define the role of homophilic interactions in IE1 transactivation, we have mapped the IE1 domains required for oligomerization. We report here that IE1 oligomerizes by a mechanism independent of enhancer binding, as demonstrated by in vitro pull-down assays using fusions of IE1 (582 residues) to the C terminus of glutathione S-transferase. In vivo oligomerization of IE1 was verified by immunoprecipitation of IE1 complexes from extracts of plasmid-transfected SF21 cells. Analyses of a series of site-directed IE1 insertion mutations indicated that a helix-loop-helix (HLH)-like domain extending from residue 543 to residue 568 is the primary determinant of oligomerization. Replacement of residues within the hydrophobic face of the putative dimerization domain disrupted IE1 homophilic interactions and caused loss of IE1 transactivation of hr-dependent promoters in plasmid transfection assays. Thus, oligomerization is required for IE1 transcriptional stimulation. HLH mutations also reduced IE1 stability and abrogated transactivation of non-hr-dependent promoters. These data support a model wherein IE1 oligomerizes prior to DNA binding to facilitate proper interaction with the symmetrical recognition sites within the hr enhancer and thereby promote the transcription of early viral genes