Mechanism of δ Mediated Transcription Activation in Bacillus subtilis: Interaction with α CTD of RNA Polymerase Stabilizes δ and Successively Facilitates the Open Complex Formation.

The α CTD (C-terminal domain of the α subunit) of RNA polymerase (RNAP) is a target for transcriptional regulators. In the transcription activation at Class I, Class II, and Class III promoters of bacteria, the transcriptional regulator, binds to DNA at different sites and interacts with the α CTD to stabilize the RNAP at the promoter or it binds to the α CTD to form a prerecruitment complex that searches for its cognate binding site. This 'simple recruitment mechanism' of the transcriptional machinery at the promoter is responsible for the activation of transcription. Strikingly, in B. subtilis the binding of RNAP at the promoter stabilizes the transcriptional regulator, δ at the -41 site of the promoter DNA through an interaction with its α CTD and successively facilitates the open complex formation. Two residues R293 and K294 of α CTD (equivalent to K297 and K298 of E. coli) are involved in the interactions with δ and essential for the activation of transcription. R293 is responsible for the stabilization of δ, while K294 is responsible for facilitating the open complex formation. Based on our data we propose a new model of transcription activation by δ of B. subtilis that is similar to (its binding location and interaction with α CTD), but distinct from (the recruitment of transcription factor by RNAP at the DNA, and enhancement of the open complex formation) the model Class II promoters in bacteria.

Characterization of C-nucleoside Antimicrobials from Streptomyces albus DSM 40763: Strepturidin is Pseudouridimycin.

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.

A Novel Function of δ Factor from Bacillus subtilis as a Transcriptional Repressor.

δ, a small protein found in most Gram-positive bacteria was, for a long time, thought to be a subunit of RNA polymerase (RNAP) and was shown to be involved in recycling of RNAP at the end of each round of transcription. However, how δ participates in both up-regulation and down-regulation of genes in vivo remains unclear. We have recently shown, in addition to the recycling of RNAP, δ functions as a transcriptional activator by binding to an A-rich sequence located immediately upstream of the -35 element, consequently facilitating the open complex formation. The result had explained the mechanism of up-regulation of the genes by δ. Here, we show that Bacillus subtilis δ could also function as a transcriptional repressor. Our results demonstrate that δ binds to an A-rich sequence located near the -35 element of the spo0B promoter, the gene involved in the regulatory cascade of bacterial sporulation and inhibits the open complex formation due to steric clash with σ region 4.2. We observed a significant increase in the mRNA level of the spo0B gene in a δ-knock-out strain of B. subtilis compared with the wild-type. Thus, the results report a novel function of δ, and suggest the mechanism of down-regulation of genes in vivo by the protein.

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, ß, ß', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator.

Promoter Escape with Bacterial Two-component σ Factor Suggests Retention of σ Region Two in the Elongation Complex.

The transition from the formation of the RNA polymerase (RNAP)-promoter open complex step to the productive elongation complex step involves "promoter escape" of RNAP. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two-component σ factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, which mimics σR4, is released gradually as transcription elongation proceeds, whereas YvrHa, which mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP.

Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation.

Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification.

Mycobacterium tuberculosis, the human pathogen that causes tuberculosis, warrants enormous attention due to the emergence of multi drug resistant and extremely drug resistant strains. RNA polymerase (RNAP), the key enzyme in gene regulation, is an attractive target for anti-TB drugs. Understanding the structure-function relationship of M. tuberculosis RNAP and the mechanism of gene regulation by RNAP in conjunction with different σ factors and transcriptional regulators would provide significant information for anti-tuberculosis drug development targeting RNAP. Studies with M. tuberculosis RNAP remain tedious because of the extremely slow-growing nature of the bacteria and requirement of special laboratory facility. Here, we have developed and optimized recombinant methods to prepare M. tuberculosis RNAP core and RNAP holo enzymes assembled in vivo in Escherichia coli. These methods yield high amounts of transcriptionally active enzymes, free of E. coli RNAP contamination. The recombinant M. tuberculosis RNAP is used to develop a highly sensitive fluorescence based in vitro transcription assay that could be easily adopted in a high-throughput format to screen RNAP inhibitors. These recombinant methods would be useful to set a platform for M. tuberculosis RNAP targeted anti TB drug development, to analyse the structure/function of M. tuberculosis RNAP and to analyse the interactions among promoter DNA, RNAP, σ factors, and transcription regulators of M. tuberculosis in vitro, avoiding the hazard of handling of pathogenic bacteria.