The bacterial enhancer-dependent RNA polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

A central event in transcription is the assembly on DNA of specific complexes near the initiation sites for RNA synthesis. Activation of transcription by one class of enhancer-binding proteins requires an RNA polymerase holoenzyme containing the specialized transcription factor, sigma-54 (sigma 54). We report here that sigma 54 alone specifically binds to promoter DNA and is responsible for many of the close contacts between RNA polymerase holoenzyme and promoter DNA, a property proposed for the major sigma 70 protein family. Binding of sigma 54 to promoter DNA is not equivalent to that of holoenzyme suggesting that there is a constraint on sigma 54 conformation when bound with core RNA polymerase. Footprints indicate sigma 54 is at the leading edge of DNA-bound holoenzyme. Like the holoenzyme, sigma 54-binding to promoter DNA does not result in DNA strand separation. Instead the specific DNA-binding activity of sigma 54 assists assembly of a closed promoter complex. This complex can be isomerized to the open (DNA melted) complex by activator protein, but promoter-bound sigma 54 alone cannot be induced to melt DNA. The pathway leading to productive transcription is similar to that proposed for eukaryotic RNA polymerase II systems.

During sporulation in Bacillus subtilis, an asymmetric division produces two cells, a forespore and mother cell, with which follow different developmental paths. The highly ordered programme of temporal and spatial gene activation during sporulation is governed by the principal RNA polymerase holoenzyme (EsigmaA) and alternative holoenzyme forms containing the developmental sigma factors sigmaH, sigmaF, sigmaE, sigmaG and sigmaK, which appear successively during development. The control mechanism(s) of temporal and selective association of multiple sigma factors with core RNA polymerase is unclear. As a first step to addressing these issues, this report quantifies the amount of each subunit of RNA polymerase that is present in the sporangium during sporulation, and analyses in vitro the relative affinities of each sigma subunit for core RNA polymerase. Using quantitative immunoblot analysis, the amounts of EsigmaA, EsigmaH, EsigmaE and EsigmaK in relation to the total amount of RNA polymerase at appropriate time-points were found to be 15%, 1%, 6% and 2%, respectively. Therefore, the core RNA polymerase is predicted to be in excess. The level of core RNA polymerase and sigmaA remained constant during the transition from vegetative growth to sporulation, whereas the sporulation-specific sigma factors appeared successively, in the order sigmaH, sigmaE and sigmaK. Competition experiments between sigma factors in an in vitro transcription system revealed the dominance of sigmaA over sigmaH and sigmaE for open promoter complex formation. These results are inconsistent with the idea that late appearing sigma factors can displace earlier appearing sigmas from the core enzyme. As the core RNA polymerase is in excess, the results suggest that successive sigma factors can bind to core RNA polymerase without having to displace earlier appearing sigma factors. Thus, the programme of gene expression during sporulation might not require mechanisms for the substitution of one sigma factor by another on the core RNA polymerase.

RNA polymerase (RNAP) is an essential and highly conserved enzyme in all organisms. The process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. In prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and RNAP an attractive target for antimicrobial agents. Our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the interaction between a bacterial RNAP and an essential sigma factor can be disrupted by introducing carefully designed mutations into sigma(A) of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant sigmas was achieved by computer-aided visualization of the RNAP-sigma interface of the B. subtilis holoenzyme (RNAP + sigma) constructed using a homology modeling approach with published crystal structures of bacterial RNAPs. Models of the holoenzyme and the core RNAP were rigorously built, evaluated, and validated. To allow a high-quality RNAP-sigma interface model to be constructed for the design of mutations, a crucial error in the B. subtilis sigma(A) sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-sigma interactions using targeted mutations.

Objectives: To analyze the Multi-Subunit (MSU) DNA dependent RNA polymerases of eubacteria and identify conserved motifs, active site regions among them and propose a plausible mechanism of action for these enzymes, using the E. coli RNA polymerase as a model system. Methods: The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNA polymerases from eubacteria. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and Site-Directed Mutagenesis (SDM) experiments and X-ray crystallographic analysis on these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Findings: Multiple Sequence Alignment (MSA) of eubacterial RNA polymerases from different bacterial sources showed only a very few highly conserved motifs among them. Possible catalytic regions in the catalytic subunits β and β‘ (in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that is reported for DNA polymerases and single subunit (SSU)DNA dependent RNA polymerases. In addition to, the invariant ‘gatekeeper/DNA template binding’ YG pair is also found in all the MSU RNA polymerases as found in all the SSU RNA polymerases and DNA polymerases. The eubacterial βsubunits are very similar in theiractive sites, catalytic regions and distance conservations between the template binding YG pair and the catalytic R, i.e., they are placed at 7 amino acids length. An invariant R is placed here at -6th position from the catalytic R, similar to SSU RNA polymerases and DNA polymerases which is proposed to play an important role in NTP selection. In the eubacterial β’ catalytic subunits, the proposed catalytic R is placed double the distance, i.e., -16 amino acids downstream from the YG pair unlike in the SSU RNA polymerasesand DNA polymerases where the distance is only 8th amino acids downstream from the YG pair. The two Ds in the completely conserved metal binding region could act as the charge shielder and orient the α-phosphates of incoming NTPs for reaction at the 3’-OH growing end. Based on the sequence analysis, a model is proposed for the initiation and elongation steps in the transcription process and a plausible mechanism of action. Applications: A comparative analysis of the bacterial and human RNA polymerases for their transcription mechanism will pave way for design new and effective drugs for many bacterial infections, including the antibiotic resistance. Keywords: Bacterial RNA Polymerases, Clustal Omega, Conserved Motifs, Multi-Subunit RNA Polymerases, Polymerase Active Sites, Polymerization Mechanism, Proofreading

The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FIIA protein, is an alternative sigma factor (sigma 28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of sigma 28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both sigma 70 RNA polymerase holoenzyme with FihD/C (E sigma 70-FlhD/C) and sigma 28 RNA polymerase holoenzyme (E sigma 28). The flhB operon could only be transcribed by E sigma 70-FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by E sigma 28 could outcompete that by E sigma 70-FlhD/C, indicating a positive autoregulation. However, E sigma 28 could not displace E sigma 70-FlhD/C bound to the fliL promoter. The sigma 28-mediated positive regulation of the class II operons involved a mechanism in which sigma 28 competed with sigma 70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the sigma 70 -10 site to the sigma 28 -10 was facilitated by formation of E sigma 70-FlhD/C pre-initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by sigma 28. We propose that class II operons may be further divided into different subcategories.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.

During bacterial transcription, sigma (σ) factors reversibly bind to RNA polymerase (RNAP) and recognize specific promoter sequences to initiate the process. While different sigma factors are utilized under different external conditions, Sigma S (RpoS, σS), a stress-responding sigma factor, is activated when bacteria face external threats. σS, which has a much lower affinity to RNAP compared with sigma D (RpoD, σ70), is controlled by a very complex network of regulatory factors. Crl protein, a transcriptional factor from Escherichia coli (E. coli, Ec), stimulates σS-dependent transcription by promoting the association of σS with core RNA polymerase. As an important regulator for σS, Crl is induced by low temperature, leading to an increased transcription rate of a subset of genes of the rpoS regulon under stress conditions or in stationary phase of growth. However, the underlying molecular mechanism for Crl/σS remains elusive. Here we describe the complete 1H, 13C and 15N chemical shift assignments of Crl as the basis for NMR structure determination and interaction studies.

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC–MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.

Modeling Transcription Initiation By Bacterial RNA Polymerase

RNA polymerase (RNAP) is the central motor of gene expression since it governs the process of transcription. In prokaryotes, this holoenzyme is formed by the RNAP core and a sigma factor. After approaching and binding the specific promoter site on the DNA, the holoenzyme-promoter complex undergoes several conformational transitions that allow unwinding and opening of the DNA duplex. Once the first DNA basepairs (∼10 bp) are transcribed in an initial transcription process, the enzyme unbinds from the promoter and proceeds downstream along the DNA while continuously opening the helix and polymerizing the ribonucleotides in correspondence with the template DNA sequence. When the gene is transcribed into RNA, the process generally is terminated and RNAP unbinds from the DNA. The first step of transcription-initiation, is considered the rate-limiting step of the entire process. This review focuses on the single-molecule studies that try to reveal the key steps in the initiation phase of bacterial transcription. Such single-molecule studies have, for example, allowed real-time observations of the RNAP target search mechanism, a mechanism still under debate. Moreover, single-molecule studies using Förster Resonance Energy Transfer (FRET) revealed the conformational changes that the enzyme undergoes during initiation. Force-based techniques such as scanning force microscopy and magnetic tweezers allowed quantification of the energy that drives the RNAP translocation along DNA and its dynamics. In addition to these in vitro experiments, single particle tracking in vivo has provided a direct quantification of the relative populations in each phase of transcription and their locations within the cell.

Fluorescence Resonance Energy Transfer (FRET) in Analysis of Transcription-Complex Structure and Function

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

In vitro transcription is now a routine biochemical technique that allows for the synthesis of RNA molecules from a DNA template. This is done utilizing a variety of bacterial and phage RNA polymerases. Transcriptional pausing is a common biological phenomenon, yet the molecular mechanism of pausing is not yet well understood. Bacterial and phage RNA polymerases have been demonstrated to behave differently when they encounter a protein blockade during transcription. The goal of this project is to characterize the transcription complex as it encounters the protein blockade. Characterization of this complex will enable us to better understand the structural and chemical interactions between the polymerase, the DNA template, and the blockade protein. The protein blockade was engineered using the dCas9 protein from the CRISPR system. By using a nuclease deficient Cas9 protein (dCas9), we can create a protein blockade that will not cleave the template The CRISPR system is an adaptive immune system found in Archaea and many Bacteria . dCas9 protein binds guide RNA that is complementary to a target DNA sequence within the transcription template sequence creating a reversible blockade at specific locations in order to study the RNA polymerase interaction. In vitro conditions for T7 RNA polymerase have been optimized, and initial characterization of the complex has been completed. This work will allow us to better understand the mechanism behind a novel, flexible, and reversible transcriptional control system.