DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37°C and results in the accumulation of an off-pathway intermediate

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild-type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus –10 region. Finally, the comparison of the results obtained at 37°C with those at 20°C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex

The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ(70) Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ(70) and Core

The N-terminal region (region 1.1) of σ(70), the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ(70) regions 2 and 4. Region 1.1 prevents the interaction of free σ(70) with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ(70)-dependent transcription from promoters that require an interaction between σ(70) region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ(70) region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ(70) region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P(uvsX). However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ(70) that lacks region 1.1 is less stable than polymerase with full-length σ(70). Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ(70) and then the AsiA/σ(70) complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ(70) plus core, yielding more free σ(70) at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ(70). Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ(70) arises through an indirect effect

Correction: Modeling HIV Vaccines in Brazil: Assessing the Impact of a Future HIV Vaccine on Reducing New Infections, Mortality and Number of People Receiving ARV

The AIDS epidemic in Brazil remains concentrated in populations with high vulnerability to HIV infection, and the development of an HIV vaccine could make an important contribution to prevention. This study modeled the HIV epidemic and estimated the potential impact of an HIV vaccine on the number of new infections, deaths due to AIDS and the number of people receiving ARV treatment, under various scenarios.The historical HIV prevalence was modeled using Spectrum and projections were made from 2010 to 2050 to study the impact of an HIV vaccine with 40% to 70% efficacy, and 80% coverage of adult population, specific groups such as MSM, IDU, commercial sex workers and their partners, and 15 year olds. The possibility of disinhibition after vaccination, neglecting medium- and high-risk groups, and a disease-modifying vaccine were also considered. The number of new infections and deaths were reduced by 73% and 30%, respectively, by 2050, when 80% of adult population aged 15-49 was vaccinated with a 40% efficacy vaccine. Vaccinating medium- and high-risk groups reduced new infections by 52% and deaths by 21%. A vaccine with 70% efficacy produced a great decline in new infections and deaths. Neglecting medium- and high-risk population groups as well as disinhibition of vaccinated population reduced the impact or even increased the number of new infections. Disease-modifying vaccine also contributed to reducing AIDS deaths, the need for ART and new HIV infections.Even in a country with a concentrated epidemic and high levels of ARV coverage, such as Brazil, moderate efficacy vaccines as part of a comprehensive package of treatment and prevention could have a major impact on preventing new HIV infections and AIDS deaths, as well as reducing the number of people on ARV. Targeted vaccination strategies may be highly effective and cost-beneficial