Andrey Feklistov

Structural Basis for Promoter −10 Element Recognition by the Bacterial RNA Polymerase σ Subunit

The key step in bacterial promoter opening is recognition of the -10 promoter element (T(-12)A(-11)T(-10)A(-9)A(-8)T(-7) consensus sequence) by the RNA polymerase σ subunit. We determined crystal structures of σ domain 2 bound to single-stranded DNA bearing-10 element sequences. Extensive interactions occur between the protein and the DNA backbone of every -10 element nucleotide. Base-specific interactions occur primarily with A(-11) and T(-7), which are flipped out of the single-stranded DNA base stack and buried deep in protein pockets. The structures, along with biochemical data, support a model where the recognition of the -10 element sequence drives initial promoter opening as the bases of the nontemplate strand are extruded from the DNA double-helix and captured by σ. These results provide a detailed structural basis for the critical roles of A(-11) and T(-7) in promoter melting and reveal important insights into the initiation of transcription bubble formation.

Bacterial Sigma Factors: A Historical, Structural, and Genomic Perspective

Transcription initiation is the crucial focal point of gene expression in prokaryotes. The key players in this process, sigma factors (σs), associate with the catalytic core RNA polymerase to guide it through the essential steps of initiation: promoter recognition and opening, and synthesis of the first few nucleotides of the transcript. Here we recount the key advances in σ biology, from their discovery 45 years ago to the most recent progress in understanding their structure and function at the atomic level. Recent data provide important structural insights into the mechanisms whereby σs initiate promoter opening. We discuss both the housekeeping σs, which govern transcription of the majority of cellular genes, and the alternative σs, which direct RNA polymerase to specialized operons in response to environmental and physiological cues. The review concludes with a genome-scale view of the extracytoplasmic function σs, the most abundant group of alternative σs.

Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center

Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2–3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351–363] proposed that rifamycins function by allosteric modulation of binding of Mg2+ to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg2+ to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNAP active center.

Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation. DOI: http://dx.doi.org/10.7554/eLife.08504.001

RNA polymerase motions during promoter melting

Trapping RNA polymerase in the act The enzyme RNA polymerase (RNAP) finds promoter elements in the genome, separates (or “melts”) the DNA strands, and transcribes the template DNA strand to give RNA. A mobile clamp in RNAP plays a key role in initiating transcription. Feklistov et al. locked the clamp of bacterial RNAP in distinct conformations by using small molecules. They then used fluorescent probes to monitor binding as the promoter DNA was separated. Unexpectedly, they found that the clamp transiently closed to nucleate DNA melting, opened to load single-stranded DNA into the active site, and then closed around the template strand to start transcription. Science, this issue p. 863 The RNA polymerase clamp closes to initiate promoter melting, opens to load melted DNA, and closes again for a tight grip on DNA. All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.

RNA polymerase: in search of promoters

Transcription initiation is a key event in the regulation of gene expression. RNA polymerase (RNAP), the central enzyme of transcription, is able to efficiently locate promoters in the genome, carry out promoter opening, and initiate RNA synthesis. All the substeps of transcription initiation are subject to complex cellular regulation. Understanding the molecular details of each step in the promoter‐opening pathway is essential for a complete mechanistic and quantitative picture of gene expression. In this minireview, primarily using bacterial RNAP as an example, I briefly summarize some of the key recent advances in our understanding of the mechanisms of promoter search and promoter opening.

Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase σ Subunit

Bacterial RNA polymerase holoenzyme relies on its σ subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the σ subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free σ, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with σ region 1 playing a role in the masking. To analyze the DNA-binding properties of the free σ, we selected single-stranded DNA aptamers that are specific to primary σ subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by σ region 2 occurs without major structural rearrangements of σ observed upon the holoenzyme formation and is not inhibited by σ regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated σ subunit and a short aptamer oligonucleotide.

Promoter recognition by bacterial alternative σ factors: the price of high selectivity?

A key step in bacterial transcription initiation is melting of the double-stranded promoter DNA by the RNA polymerase holoenzyme. Primary sigma factors mediate the melting of thousands of promoters through a conserved set of aromatic amino acids. Alternative sigmas, which direct transcription of restricted regulons, lack the full set of melting residues. In this issue of Genes & Development, Koo and colleagues (pp. 2426-2436) show that introducing the primary sigma melting residues into alternative sigmas relaxes their promoter specificity, pointing to a trade-off of reduced promoter melting capacity for increased promoter stringency.

Promoter melting by an alternative σ, one base at a time

Housekeeping σ factors are initiation factors for the bacterial RNA polymerase at most promoters, whereas alternative σs direct focused responses to specific environmental conditions. Structural and functional analysis of an alternative σ complexed with its cognate −10 motif elucidates the mechanism for initiation of strand opening, highlighting two critical properties: why alternative σs, compared to housekeeping σs, recognize so few promoters and how their promoter-recognition strategy was diversified during evolution.

Site-specific aptamer inhibitors of Thermus RNA polymerase

Bacterial RNA polymerase (RNAP) is an RNA-synthesizing molecular machine and a target for antibiotics. In transcription, RNAP can interact with DNA sequence-specifically, during promoter recognition by the σ-containing holoenzyme, or nonspecifically, during productive RNA elongation by the core RNAP. We describe high-affinity single-stranded DNA aptamers that are specifically recognized by the core RNAP from Thermus aquaticus. The aptamers interact with distinct epitopes inside the RNAP main channel, including the rifamycin pocket, and sense the binding of other RNAP ligands such as rifamycin and the σA subunit. The aptamers inhibit RNAP activity and can thus be used for functional studies of transcription and development of novel RNAP inhibitors.