RNA Polymerase-promoter Open Complex Research Articles

Clamp Interactions with +3/+6 Duplex and Upstream-to-Downstream Allosteric Effects in Late Steps of Forming a Stable RNA Polymerase-Promoter Open Complex

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λPR and T7A1 promoters form at similar rates but have very different lifetimes. To understand the downstream interactions responsible for OC lifetime, how promoter sequence directs them and when they form, we report lifetimes of stable OC and unstable late (I2) intermediates for promoters with different combinations of λPR (L) and T7A1 (T) discriminators, core promoters and UP elements. I2 lifetimes are similarly short, while stable OC lifetimes differ greatly, determined largely by the discriminator and modulated by core-promoter and UP elements. The free energy change ΔG3o for I2 → stable OC is approximately −4 kcal more favorable for L-discriminator than for T-discriminator promoters. Downstream-truncation at +6 (DT+6) greatly destabilizes OC at L-discriminator but not T-discriminator promoters, making all ΔG3o values similar (approximately −4 kcal). Urea reduces OC lifetime greatly by affecting ΔG3o. We deduce that urea acts by disfavoring coupled folding of key elements of the β’-clamp, that I2 is an open-clamp OC, and that clamp-closing in I2 → stable OC involves coupled folding. Differences in ΔG3o between downstream-truncated and full-length promoters yield contributions to ΔG3o from interactions with downstream mobile elements (DME) including β-lobe and β’-jaw, more favorable for L-discriminator than for T-discriminator promoters. We deduce how competition between far-downstream DNA and σ70 region 1.1 affects ΔG3o values. We discuss variant-specific ΔG3o contributions in terms of the allosteric network by which differences in discriminator and −10 sequence are sensed and transmitted downstream to affect DME-duplex interactions in I2 → stable OC.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example.

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

Open complex DNA scrunching: A key to transcription start site selection and promoter escape.

Bacterial RNA polymerase-promoter open complexes can exist in a range of states in which the leading edge of the enzyme moves but the trailing edge does not, a phenomenon we refer to as "open complex scrunching." Here we describe how open complex scrunching can determine the position of the transcription start site for some promoters, modulate the level of expression, and potentially could be targeted by factors to regulate transcription. We suggest that open complex scrunching at the extraordinarily active ribosomal RNA promoters might have evolved to initiate transcription at an unusual position relative to the core promoter elements in order to maximize the rate of promoter escape.

Large Effects of Discriminator Exchanges on the RNA Polymerase-Promoter Open Complex Structure, Lifetime and Transcription Initiation Patterns

Escherichia coli RNA polymerase holoenzyme (RNAP; consisting of α2ββ’ω + σ70 subunits) is the molecular machine of transcription. This machinery is set in motion by initial recognition of −35 and −10 regions of linear promoter DNA by σ70 and of the UP element region by the two flexibly-tethered α-CTDs. A series of large conformational changes in both RNAP and DNA, driven by binding free energy, bend the downstream duplex into the active site cleft and open 13 base pairs including the −10 and discriminator regions and the transcription start site (+1). Subsequently at some promoters the initial unstable (lifetime 1 s) open complex is stabilized by a network of interactions involving the discriminator DNA, σ70 region 1.1, and downstream mobile elements (DME) of RNAP. These increase open complex lifetime to 6 minutes for T7A1 promoter and 17 hrs for the λPR promoter. An important but unanswered question is how the stability of the open complex affects its ability to initiate RNA synthesis upon binding of NTPs. Here we address this question by comparing lifetimes, structural features, and patterns of short and long RNA synthesized from λPR promoter with either the λPR or T7A1 discriminator, using nitrocellulose filter binding assays, permanganate footprinting and transcription assays. We also compare lifetimes and structural features of λPR , T7A1 and ribosomal rrnBP1 promoters with either λPR or T7A1 discriminator, as well as examine abortive and productive transcription products by acrylamide gel and novel mass spectrometric analysis to determine the extent to which the discriminator region is responsible for open complex lifetime, and to elucidate its effects on transcription initiation. This work is supported through funding by the NIH (GM103061).

Upstream promoter sequences and αCTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex

We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.

Probing the Role of Region 2 of Escherichia coli σ70 in Nucleation and Maintenance of the Single-Stranded DNA Bubble in RNA Polymerase-Promoter Open Complexes

Publisher Summary This chapter reviews the bacterial RNA polymerase (RNAP), referred to as the “holoenzyme,” or “Eσ,” is a complex of the “core enzyme” (E) and sigma factor (σ), a type of bacterial initiation factor. The multisubunit core enzyme shows similarities in both sequence and structure to the multisubunit eukaryotic RNAP. The role of the sigma factor is to recognize promoter DNA and to initiate the process of RNAP-induced strand separation to form the transcription-competent open promoter complex. The chapter discusses the major advances in the understanding of transcriptional apparatus achieved with the determination of the structure of the bacterial sigma factor, core enzyme, holoenzyme, and a cocomplex of holoenzyme, with a forked DNA encompassing most of the sequence recognized by RNAP. Relevant features of these structures include: (1) the large total area of contact between core and σ 70 , (2) DNA-binding groups of sigma factor that stick out from the body of the holoenzyme, and (3) the existence of separate tunnels for each strand of promoter DNA. This leads the template strand deep in the hydrophobic core of the RNAP to the active site of the enzyme while keeping the nontemplate strand much closer to the surface.

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

Publisher Summary This chapter discusses the Fluorescence Resonance Energy Transfer (FRET) to monitor movement of RNA polymerase (RNAP) relative to DNA during transcription and to define three-dimensional structures of transcription complexes in solution. FRET is a physical phenomenon that permits measurement of distances. FRET occurs in a system where a fluorescent probe serves as a donor and a second fluorescent probe serves as an acceptor, where the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. The chapter presents protocols for FRET experiments that permit measurement of distances between positions on upstream DNA and positions within RNAP (“trailing-edge FRET”), distances between positions on downstream DNA and positions within RNAP (“leading-edge FRET”), and distances between positions on RNAP core and positions within σ70 (“core- σ70FRET”). Later the preparation of DNA fragments, sigma σ70, RNAP core, RNAP holoenzyme, and RNA polymerase-promoter open complex (RPo) is discussed. DNA–RNAP FRET permits monitoring of movement of RNAP relative to DNA during promoter escape and elongation and provides information about three-dimensional structures of RPo and RDe. The approach involves two complementary sets of experiments: trailing-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP and a fluorescent probe on DNA upstream of RNAP; and leading-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP, and a fluorescent probe on DNA downstream of RNAP.

Structural Organization of Bacterial RNA Polymerase Holoenzyme and the RNA Polymerase-Promoter Open Complex

We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding σ 70-(RNA polymerase core), σ 70-DNA, and σ 70-RNA interactions. The positions of σ 70 regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of σ 70 region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a “molecular mimic” of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.

Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex

Formation of an initiation-competent RNA polymerase-promoter complex involves DNA melting over a region of about 12 base-pairs, which includes the start site of transcription, thus enabling the template strand to base-pair with the initiating nucleoside triphosphates. By studying the effects of alanine substitutions, we have investigated the role of the aromatic amino residues in the Escherichia coli σ 70 conserved region 2.3 in promoter strand separation. The resulting mutants were assessed for their activity in vivo in the context of a σ 70/σ 32 hybrid sigma factor that could be targeted to a specific hybrid promoter in the cell. All substitutions lead to an at least twofold reduction in expression of the hybrid promoter-driven reporter gene. The in vitro assay of single substitutions indicated cold sensitivity similar to that previously observed with analogous substitutions in Bacillus subtilis σ A. Kinetic assays showed that these substitutions slowed the rate of open complex formation at 37°C as well. RNA polymerase reconstituted with a σ 70 containing multiple alanine substitutions readily binds to promoter DNA, but then proceeds slowly beyond the first intermediate complex on the pathway to formation of the transcription-competent complex. These data demonstrate that together the aromatic residues in region 2.3 of E. coli σ 70 ensure that DNA strand separation proceeds efficiently, even if no individual residue may be essential for accomplishment of the process.

Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria

We have identified a mutant in RPB3, the third-largest subunit of yeast RNA polymerase II, that is defective in activator-dependent transcription, but not defective in activator-independent, basal transcription. The mutant contains two amino-acid substitutions, C92R and A159G, that are both required for pronounced defects in activator-dependent transcription. Synthetic enhancement of phenotypes of C92R and A159G, and of several other pairs of substitutions, is consistent with a functional relationship between residues 92-95 and 159-161. Homology modeling of RPB3 on the basis of the crystallographic structure of alphaNTD indicates that residues 92-95 and 159-162 are likely to be adjacent within the structure of RPB3. In addition, homology modeling indicates that the location of residues 159-162 within RPB3 corresponds to the location of an activation target within alphaNTD (the target of activating region 2 of catabolite activator protein, an activation target involved in a protein-protein interaction that facilitates isomerization of the RNA polymerase promoter closed complex to the RNA polymerase promoter open complex). The apparent finding of a conserved surface required for activation in eukaryotes and bacteria raises the possibility of conserved mechanisms of activation in eukaryotes and bacteria.

DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA

Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Eσ70 RNA polymerase (R) and the λPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0°C, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends ∼30 bases downstream and at least ∼40 bases upstream from the start site. I1 has a short lifetime (⩽15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37°C to 0°C, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0°C.Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Eσ70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0°C, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k−2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures.

A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes.

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.

Site-directed chemical modification for probing DNA-protein interactions: Osmium tetroxide modification of the −10 site of the lacUV5 promoter enhances open complex formation

A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.

Helix stability in prokaryotic promoter regions.

Prokaryotic promoters have been extensively studied to relate sequence features to promoter function. Here we examine the relationship between double-helix stability and promoter activity. The double-helix stability is evaluated from sequence data by free energy computation, based on reported values of dinucleotide free energies for strand separation. For a collection of 168 promoters, we find that within a 500-nucleotide span around the transcription initiation site the -10 region is the least stable. There is no correlation between the free energies and the rates of RNA polymerase-promoter open complex formation measured for 25 promoters. We also compare the free energies of 121 promoter mutations across the -35 and -10 consensus regions with the free energies of the corresponding wild-type sequences. These pairwise mutant-wild-type comparisons provide a particularly good test since the examined sequences differ only in one nucleotide so that all other sequence-dependent effects remain the same. About 80% of the mutations in the -10 region that show increased/reduced promoter activity are less/more stable than the wild types. The observed high free energy peak and the mutation data strongly support the conjecture that the instability, or melting properties, of the -10 region plays a significant role in promoter function.

Deletion analysis of RNA polymerase interaction sites in the Escherichia coli lactose operon regulatory region

Two sets of deletions produced in vitro by S1 nuclease were used to study the structure and function of promoters lacPUV5 and lacP115. The upstream boundary of the RNA polymerase binding site in lacPUV5 was determined by comparing the levels of beta-galactoside expression in vivo programmed from a set of deletions progressively extending into the -35 region of the lacPUV5 promoter. Sequences upstream from base-pair -37 are not necessary for the full functioning of lacPUV5. A deletion that removes base-pair -37 retains only half of the promoter activity. Deletion of the first T X A base-pair of the consensus -35 region sequence, 5' T-T-T-A-C-A 3', leads to a sixfold reduction of promoter activity. Deletion of the whole -35 region of lacPUV5 leads to at least a 20-fold reduction of its promoter activity. Abortive initiation assays were performed on the fully functional lacPUV5 and two lacPUV5 deletions, which removed part of the -35 consensus sequence, to study their effect on the kinetics of RNA polymerase-promoter open complex formation. These two deletions show a 3.5 to 7-fold reduction in KB. Analysis in vivo of lacP115 showed that sequence information upstream from the -35 region is important for the full functioning of lacP115. A deletion removing sequences upstream from -41 caused a three- to fourfold reduction in promoter activity, apparently due to reduced transcription initiation. lacP115 has a much lower k2 value than lacPUV5; its KB value is about threefold higher than that of lacPUV5.

Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions.

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.