Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.

Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence.

Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined by the ability of the elongating RNA polymerase to recognize the sequence of the RNA-DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly 'senses' the shape and/or identity of base pairs of the RNA-DNA hybrid. Recognition of the RNA-DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through 'in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA-DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation.

The σ70 region 1.2 regulates promoter escape by unwinding DNA downstream of the transcription start site.

The mechanisms of abortive synthesis and promoter escape during initiation of transcription are poorly understood. Here, we show that, after initiation of RNA synthesis, non-specific interaction of σ(70) region 1.2, present in all σ(70) family factors, with the non-template strand around position -4 relative to the transcription start site facilitates unwinding of the DNA duplex downstream of the transcription start site. This leads to stabilization of short RNA products and allows their extension, i.e. promoter escape. We show that this activity of σ(70) region 1.2 is assisted by the ß-lobe domain, but does not involve the ß'-rudder or the ß'-switch-2, earlier proposed to participate in promoter escape. DNA sequence independence of this function of σ(70) region 1.2 suggests that it may be conserved in all σ(70) family factors. Our results indicate that the abortive nature of initial synthesis is caused, at least in part, by failure to open the downstream DNA by the ß-lobe and σ region 1.2.

Tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex.

Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

Stepwise mechanism for transcription fidelity.

BACKGROUND: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. RESULTS: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH. CONCLUSIONS: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.