Downstream Duplex Research Articles

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes.

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA

SummaryG-quadruplexes (G4s) are non-canonical DNA structures with critical roles in DNA metabolisms. To resolve those structures that can cause replication fork stalling and genomic instability, single-stranded DNA-binding proteins and helicases are required. Here, we characterized the interplay between RPA and helicases on G4s using single-molecule FRET. We first discovered that human RPA efficiently prevents G4 formation by preempting ssDNA before its folding. RPA also differentially interacts with the folded G4s. However, helicases such as human BLM and yeast Pif1 have different G4 preferences from RPA mainly based on loop lengths. More importantly, both RPA and these helicases are required for the stable G4 unfolding, as RPA promotes helicase-mediated repetitive unfolding into durative linear state. Furthermore, BLM can traverse G4 obstacles temporarily disrupted by RPA and continue to unwind downstream duplex. We finally proposed the mechanisms underlying above functions of RPA in preventing, resolving, and assisting helicases to eliminate G4s.

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex.

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3-Cy5 distance to ∼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper β'-clamp while downstream DNA contacts the top of the β-pincer in I1E. Converting I1E to I1M (∼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on β') away from downstream DNA (on β) to increase the Cy3-Cy5 distance by ∼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.

Effects of Discriminator Changes on Open Complex Formation, Stabilization, and Transcription Initiation

The discriminator is the region of promoter DNA between the key −10 recognition region for RNA polymerase (RNAP) and the transcription start site (TSS). The length, sequences of the discriminator, and the interactions between RNAP mobile elements and promoter DNA the discriminator direct, are major determinants of the lifetime and the structure of the RNAP-promoter open complex (OC). The OC lifetime and rate of formation can both vary by ∼4 orders of magnitude for different promoter sequences, and together regulate the rate of initiation and escape of RNAP from these promoters. We are investigating a series of discriminators including those of the long-lived λPR OC (GGTTGC) and the short-lived rrnBP1 OC (GCGCCACC), in the context of the upstream λPR promoter and a modified λPR transcribed region. Fast kinetic methods with filter binding detection are used to determine overall rate constants for open complex formation and dissociation, and to dissect contributions to these rate constants from initial equilibria involving closed or open complexes (CC or OC), and the rate-determining isomerizations step between them. Our results to date indicate that the discriminator affects primarily the steps that stabilize the initial OC, and has only limited effects on the DNA closing rate constant or on the overall second order rate constant for OC formation. From previous studies, interactions of in-cleft (ss) DNA and downstream (ds) DNA with RNAP change in the process of stabilization of the initial OC. Regions 1.1 and 1.2 of σ70 and downstream mobile elements of core RNAP are involved, and the orientation of the downstream duplex is changed. We are investigating these interactions and conformational changes for different discriminators in more detail, and the relationship to initiation and promoter escape of RNAP.

Template and primer requirements for DNA Pol θ-mediated end joining

DNA Pol θ-mediated end joining (TMEJ) is a microhomology-based pathway for repairing double-strand breaks in eukaryotes. TMEJ is also a pathway for nonspecific integration of foreign DNAs into host genomes. DNA Pol θ shares structural homology with the high-fidelity replicases, and its polymerase domain (Polθ) has been shown to extend ssDNA without an apparent template. Using oligonucleotides with distinct sequences, we find that with Mg2+ and physiological salt concentrations, human Polθ has no terminal transferase activity and requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for DNA synthesis. Polθ can tolerate a mismatched base pair at the primer end but loses >90% activity when the mismatch is 2 bp upstream from the active site. Polθ is severely inhibited when the template strand has a 3' overhang within 3-4 bp from the active site. In line with its TMEJ function, Polθ has limited strand-displacement activity, and the efficiency and extent of primer extension are similar with or without a downstream duplex.

Bending and Wrapping of Upstream Promoter DNA on E. coli RNA Polymerase Facilitates Open Complex Formation in Transcription Initiation; A Fluorescence (FRET, PIFE) Study

Removal of the far‐upstream region of promoter DNA reduces the isomerization rate of closed complexes (CC) to open complexes (OC) of Escherichia coli RNA polymerase (RNAP) and λPR DNA by 1–2 orders of magnitude. Upstream DNA is proposed to facilitate OC formation by wrapping on RNAP and remodeling the downstream cleft to allow bending of the downstream duplex into the cleft prior to opening. Here we report stopped flow fluorescence kinetic studies of open complex formation at λPR promoter DNA labeled at far‐upstream and downstream positions with Cy3 and/or Cy5 fluorescent probes. We determine the time course of forming the CC ensemble (10 ms – 3 s at 19 C), in which the upstream and downstream duplex DNA are bent and wrapped on RNAP. Cy3 to Cy5 FRET (fluorescence resonance energy transfer) detects wrapping, and single‐dye PIFE (protein‐induced fluorescence enhancement) detects transient contacts of the far‐upstream DNA with the downstream cleft, and contacts of the downstream DNA as it is bent into the cleft. Subsequently (3 s – 400 s) we determine the time course of the isomerization of the CC ensemble to OC, in which the upstream DNA remains bent and wrapped but where contacts of the upstream DNA with the downstream cleft are released. These results are consistent with the model for facilitation of isomerization by wrapping of upstream DNA, and with previous footprinting and kinetic information.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination

Eukaryotic DNA polymerase of the polX family, such as pol μ and terminal deoxynucleotidyl transferase (TdT), are key components of the non-homologous end-joining or V(D)J recombination machinery, respectively. The established role of TdT is to add random nucleotides during V(D)J recombination. Here we show that TdT also has a templated-polymerase activity, similar to pol μ, in the presence of higher concentrations of a downstream DNA duplex, and performs a micro-homology single base-pair search to align the DNA synapsis. To understand the molecular basis of this alignment, we solve crystal structures of TdT with four DNA strands and study the influence ofthe 3' protruding end. Two mutations in TdT inspired by sequence alignments with pol μ further improve the templated activity. We propose that both templated and untemplated activities of TdT are needed to explain the distributions of lengths of N regions observed experimentally in Tcell receptors and antibodies.

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.

Systematic Studies of Effects of the Discriminator on Stabilization of the Open Complex and Abortive and Productive Initiation

E. coli RNA polymerase (RNAP) is a large molecular machine (subunits: αββ′ω and specificity factor σ70) with many moving parts that binds to and opens duplex promoter DNA to initiate transcription. These events are set in motion by initial interactions between σ70 and the −35 and −10 elements of the promoter, and between the flexibly tethered α‐CTDs and the UP element of the promoter. Binding free energy drives a series of conformational changes that culminate in opening of 13 base pairs between the −10 region and the start site, forming an initial unstable open complex, which is stabilized at some promoters by a network of interactions involving the discriminator and σ70 region 1 in the cleft, and between the downstream duplex and downstream mobile elements (DME) of RNAP. Different promoter open complexes differ greatly in lifetime: the open complex at the rrnB P1 ribosomal promoter is very short‐lived (~1 s), as compared to 6 minutes for T7A1, and 17 hours for λPR promoter. To investigate effects of discriminator sequence and length on the structure, lifetime and function of the open complex, we are exchanging discriminator regions between promoters and determining KMnO4 reactivities of open thymine bases, dissociation rate constants and patterns of abortive and productive initation using a common initial transcribed sequence. We extend previous findings by observing that the discriminator can be the primary sequence determinant of open complex lifetime; for example, inserting the T7A1 or rrnB P1 discriminator into λPR reduces its lifetime nearly to that of these promoters, while inserting the λPR discriminator into T7A1 increases its lifetime nearly to that of λPR. Footprinting reveals that template strand thymines upstream of the start site in the longer lived complexes have greater KMnO4 reactivity. To interpret this, we propose that RNAP binds the phosphate backbone of this strand; tighter binding unstacks the bases more, as shown for SSB‐ssDNA by Lohman, and hence increases their KMnO4 reactivity. To address the question of how the discriminator sequence and open complex lifetime affect abortive and productive initiation, we are conducting single round transcription assays on series of promoters differing only in the discriminator region. For the λPR series of discriminator variants, reproducible differences are observed in the amounts and pattern of both short and long products. Currently, research is focused on variants that exchange the λPR WT discriminator with those of rrnBP1 and T7A1 to determine how the discriminator region affects the distribution of short and long products as well as the absolute amount of products produced.

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).

Kinetics and Mechanism of Formation and Stabilization of the RNA Polymerase-Promoter Open Compex

Specific binding of E. coli RNA polymerase holoenzyme (RNAP; α2ββ’σ70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the −10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Forster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061).

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >105-fold and >102-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ70 region 1.1 (σ1.1), β′ jaw and β′ sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases −4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either β′ jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.

Initial events in bacterial transcription initiation.

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the −10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase.

The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH–NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.

Coupling of Downstream RNA Polymerase–Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex

Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP–promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP–downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.

Characterization of Transcription Initiation Intermediates in Escherichia Coli RNA Polymerase by Fluorescence

Transcription initiation involves a series of conformational changes that are triggered in both RNA polymerase (RNAP) and DNA after recognition of −35, −10 elements of promoter DNA by RNAP holoenzyme, consisting of catalytic core and a specificity factor, σ. RNA polymerase (RNAP) functions as a molecular isomerization machine, binding and bending the promoter DNA in the initial closed complex and using binding free energy to melt 13 bp of DNA in the active site cleft to form an initial unstable open promoter complex, which is converted to a final open complex (RPo) (Saecker et al, '11). Promoter melting is thought to commence upon flipping of −11A of the non-template strand out of the DNA helix (Schroeder et al, '09) but several questions remain open. After recruitment of RNAP to promoter DNA, what conformational changes occur in the complex to put the downstream DNA duplex in the cleft? When and how is the DNA bent and wrapped around RNAP? When does the −11A element rotate vis-a-vis wrapping? To answer these questions, we are characterizing early intermediates at the λPr promoter using equilibrium and kinetic bulk fluorescence measurements. Fluorescence Resonance Energy Transfer (FRET) experiments monitor the extent of wrapping whereas stopped-flow “Beacon” assays provide information about −11A base-flipping and opening of −10 region (Mekler et al, '11). FRET experiments show that promoter DNA is wrapped around RNAP in the advanced closed complex and that wrapping persists in open complexes. The wrapped open complex is found to be transcriptionally competent. Real-time stopped-flow kinetic experiments are in progress to determine the sequence of steps in which upstream and downstream duplex promoter DNA are bent toward and wrapped around RNAP to put the start site region in the cleft and open it.