Dynamics and logic of promoter melting.

Despite significant progress in our understanding of promoter melting dynamics, underlying principles of the process remain elusive, with opposing views on key aspects held by many in the field. Here, I discuss the mechanistic logic behind the interplay of thermal and deterministic forces acting to create transcriptionally competent promoter complexes.

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.

6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase.

Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ70-holoenzyme (Eσ70) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ70 determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ70 and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.

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.

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.

Promoter recognition by bacterial alternative sigma 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.

Single-strand promoter traps for bacterial RNA polymerase.

Besides canonical double-strand DNA promoters, multisubunit RNAPs (RNA polymerases) recognize a number of specific single-strand DNA and RNA templates, resulting in synthesis of various types of RNA transcripts. The general recognition principles and the mechanisms of transcription initiation on these templates are not fully understood. To investigate further the molecular mechanisms underlying the transcription of single-strand templates by bacterial RNAP, we selected high-affinity single-strand DNA aptamers that are specifically bound by RNAP holoenzyme, and characterized a novel class of aptamer-based transcription templates. The aptamer templates have a hairpin structure that mimics the upstream part of the open promoter bubble with accordingly placed specific promoter elements. The affinity of the RNAP holoenzyme to such DNA structures probably underlies its promoter-melting activity. Depending on the template structure, the aptamer templates can direct synthesis of productive RNA transcripts or effectively trap RNAP in the process of abortive synthesis, involving DNA scrunching, and competitively inhibit promoter recognition. The aptamer templates provide a novel tool for structure-function studies of transcription initiation by bacterial RNAP and its inhibition.

Highly specific aptamer trap for extremophilic RNA polymerases.

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus phylum. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.

Crystallographic analysis of an RNA polymerase σ-subunit fragment complexed with -10 promoter element ssDNA: quadruplex formation as a possible tool for engineering crystal contacts in protein-ssDNA complexes.

Structural studies of -10 promoter element recognition by domain 2 of the RNA polymerase σ subunit [Feklistov & Darst (2011), Cell, 147, 1257-1269] reveal an unusual crystal-packing arrangement dominated by G-quartets. The 3'-terminal GGG motif of the oligonucleotide used in crystallization participates in G-quadruplex formation with GGG motifs from symmetry-related complexes. Stacking between neighboring G-quadruplexes results in the formation of pseudo-continuous four-stranded columns running throughout the length of the crystal (G-columns). Here, a new crystal form is presented with a different arrangement of G-columns and it is proposed that the fortuitous finding of G-quartet packing could be useful in engineering crystal contacts in protein-ssDNA complexes.

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 Mg(2+) 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 Mg(2+) 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 Mg(2+) to the RNAP active center.

RNA polymerase motions during promoter melting.

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.

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 σ(A) dissociation.

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 sigma subunit.

Bacterial RNA polymerase holoenzyme relies on its sigma subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the sigma subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free sigma, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with sigma region 1 playing a role in the masking. To analyze the DNA-binding properties of the free sigma, we selected single-stranded DNA aptamers that are specific to primary sigma 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 sigma region 2 occurs without major structural rearrangements of sigma observed upon the holoenzyme formation and is not inhibited by sigma 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 sigma subunit and a short aptamer oligonucleotide.

A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme.

During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, sigma-subunit and GreB.

Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs. The process of transcription is accompanied by complex structural rearrangements of RNAP. Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP. Using in vitro selection methods, we obtained 13 classes of ssDNA aptamers against Escherichia coli core RNAP. The minimal nucleic acid scaffold (an oligonucleotide construct imitating DNA and RNA in elongation complex), rifampicin and the sigma70-subunit inhibited binding of the aptamers to RNAP core but did not affect the dissociation rate of preformed RNAP-aptamer complexes. We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel. In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP-aptamer complexes. This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP-aptamer complexes by inducing conformational changes in the channel. We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investigating the conformational flexibility of the enzyme.