Open Promoter Complex Research Articles

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.

Cooperativity and Interaction Energy Threshold Effects in Recognition of the −10 Promoter Element by Bacterial RNA Polymerase

RNA polymerase (RNAP) melts promoter DNA to form transcription-competent open promoter complex (RPo). Interaction of the RNAP σ subunit with non-template strand bases of a conserved −10 element (consensus sequence T-12A-11T-10A-9A-8T-7) is an important source of energy-driving localized promoter melting. Here, we used a fluorometric RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with −10 element nucleotides. The data reveal a strong cooperation between RNAP interactions with individual −10 element non-template strand nucleotides and indicate that recognition of the −10 element bases occurs only when the overall RNAP −10 element binding acquires a free energy below a ca. −3 kcal/mol threshold. These results may be explained by that the individual interactions between RNAP and −10 element nucleotides cooperatively contribute into retaining a characteristic recognizable conformation of the −10 element nt-strand that was revealed in recent structural studies. The RNAP interaction with T/A-12 base pair was found to be strongly stimulated by RNAP interactions with other −10 element bases and with promoter spacer between the −10 and −35 promoter elements. The data also indicate that unmelted −10 promoter element can impair RNAP interactions with promoter DNA upstream of the −11 position. We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of transcription initiation complex formation.

Subunit Dynamics and Nucleotide-Dependent Asymmetry of an AAA+ Transcription Complex

Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA+ protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA+ domain of one well-studied bEBP in complex with its substrate, the σ54 subunit of RNA polymerase. Our results demonstrate that the free AAA+ domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ54 binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs.

Structure and function of CarD, an essential mycobacterial transcription factor

CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.

Cooperativity and interaction energy threshold effects in recognition of the −10 promoter element by bacterial RNA polymerase

RNA polymerase (RNAP) melts promoter DNA to form transcription-competent open promoter complex (RPo). Interaction of the RNAP σ subunit with non-template strand bases of a conserved −10 element (consensus sequence T−12A−11T−10A−9A−8T−7) is an important source of energy-driving localized promoter melting. Here, we used an RNAP molecular beacon assay to investigate interdependencies of RNAP interactions with −10 element nucleotides. The results reveal a strong cooperation between RNAP interactions with individual −10 element non-template strand nucleotides and indicate that recognition of the −10 element bases occurs only when free energy of the overall RNAP −10 element binding reaches a certain threshold level. The threshold-like mode of the −10 element recognition may be related to the energetic cost of attaining a conformation of the −10 element that is recognizable by RNAP. The RNAP interaction with T/A−12 base pair was found to be strongly stimulated by RNAP interactions with other −10 element bases and with promoter spacer between the −10 and −35 promoter elements. The data also indicate that unmelted −10 promoter element can impair RNAP interactions with promoter DNA upstream of the −11 position. We suggest that cooperativity and threshold effects are important factors guiding the dynamics and selectivity of RPo formation.

Global Regulation of Promoter Melting in Naive Lymphocytes

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.

An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity

The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the σ-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively ‘RPi’). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ54-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

The X‐ray Crystal Structure of Escherichia Coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the best‐studied bacterial RNAP and has been used as the model RNAP for potential RNAP‐targeting antibiotic screening and evaluation. However, the X‐ray crystal structure of E. coli RNAP has been limited to individual domains. In this study, E. coli RNAP core enzyme (E) and holoenzyme (Eσ70) were prepared from overexpressed proteins. The holoenzyme was crystallized and solved its X‐ray structure at 3.8 Å resolution. The E. coli holoenzyme structure shows the σ region 1.1 (σ1.1) and α subunit C‐terminal domain (αCTD) structures for first time in any bacterial RNAP. The structural comparison between the E. coli and Thermus RNAPs shows that the ω subunit structure is different in E. coli from that in Thermus that may be related to the fact that the only E. coli RNAP is able to respond ppGpp for transcription regulation. The σ1.1 is positioned at the RNAP DNA binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that the σ1.1 contains a basic patch on its surface, which may play an important role in the DNA interaction to facilitate the transition from the closed to open promoter complexes. The αCTD is positioned next to the σ domain 4 (σ4) with a fully stretched linker between the N‐ and C‐terminal domains. E. coli RNAP crystals can be conveniently prepared from overexpressed proteins, allowing further structural studies of bacterial RNAP mutants, including antibiotic resistant RNAPs.

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ(70) holoenzyme, which shows σ region 1.1 (σ1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.

Structural visualization of key steps in human transcription initiation.

Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. We have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE, and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed PIC and the open-promoter complex and regulate start site selection. Comparison of open versus closed pre-initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, supports a DNA translocation model of XPB and explains its essential role in promoter opening.

Regulation of Initiation and Transcription by E. coli RNA Polymerase Sigma Region 1.2 and Promoter Sequence

Although much progress has been made in the study of transcription initiation by E. coli RNA polymerase (RNAP), many questions still remain regarding the “isomerization” steps which open the DNA and stabilize the transcription-competent open promoter complex. Large scale conformational changes in RNAP and promoter DNA occur, and allosteric communication occurs over large distances, but the details are poorly understood. are these mechanistic steps universal or promoter-specific? How do they relate to transcriptional output? To address these questions, we are determining the kinetics of transcription initiation and transcription product distribution at the model bacteriophage promoters T7A1 and ΔPR, which exhibit very different kinetic and transcriptional behavior, as well as several variants of both. These promoter sequences differ greatly in the “discriminator” region between the −10 element and the transcription start site. The nontemplate strand of the discriminator region is known to interact with region 1.2 of the RNAP σ subunit, stabilizing the open promoter complex. Our results show that this interaction is coupled to the assembly of RNAP downstream mobile elements (DME) on duplex promoter DNA. Together, σ 1.2-discriminator interactions and DME assembly increase lifetimes of open complexes up to 105-fold and control the RNA product distribution (i.e., short abortives versus long transcripts). The DME are also targets of regulation by proteins such as T7 gp2, and are proposed to be involved in interactions between RNAP and far upstream DNA which affect the kinetics of putting the downstream duplex in the active site cleft and opening it. These studies show that discriminator interactions with σ 1.2 control the structure, lifetime, and transcriptional outputs of the final open complex, suggesting that the discriminator region may be critically important for transcription regulation in the cell.

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

RNA polymerase III subunit architecture and implications for open promoter complex formation

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization.

Gene silencing by H‐NS from distal DNA site

In the modern concept of gene regulation, 'DNA looping' is the most common underlying mechanism in the interaction between RNA polymerase (RNAP) and transcription factors acting at a distance. This study demonstrates an additional mechanism by which DNA-bound proteins communicate with each other, by analysing the bacterial histone-like nucleoid-structuring protein (H-NS), a general transcriptional silencer. The LEE5 promoter (LEE5p) of enteropathogenic Escherichia coli was used as a model system to investigate the mechanism of H-NS-mediated transcription repression. We found that H-NS represses LEE5p by binding to a cluster of A tracks upstream of -114, followed by spreading to a site at the promoter through the oligomerization of H-NS molecules. At the promoter, the H-NS makes a specific contact with the carboxy terminal domain of the α subunit of RNAP, which prevents the processing of RNAP-promoter complexes into initiation-competent open promoter complexes, thereby regulating LEE5p from distance.

Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised

The Escherichia coli-infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the E. coli RNAp is the β′ jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β′ jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β′ jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β′ jaw. The 7009 E. coli strain is resistant to T7 because it carries a charge reversal point mutation in the β′ jaw that prevents Gp2 binding. However, a T7 phage encoding a mutant form of Gp2, called Gp2β, which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2β, we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β′ jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.

A dual switch controls bacterial enhancer-dependent transcription.

Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ54 factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation.

Fidaxomicin Is an Inhibitor of the Initiation of Bacterial RNA Synthesis

Fidaxomicin was recently approved for the treatment of Clostridium difficile infection. It inhibits transcription by bacterial RNA polymerase. Because transcription is a multistep process, experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step. DNA footprinting experiments were also conducted to further elucidate the stage inhibited. Fidaxomicin blocks initiation only if added before the formation of the “open promoter complex,” in which the template DNA strands have separated but RNA synthesis has not yet begun. Binding of fidaxomicin precludes the initial separation of DNA strands that is prerequisite to RNA synthesis. These studies show that it has a mechanism distinct from that of elongation inhibitors, such as streptolydigin, and from the transcription initiation inhibitors myxopyronin and the rifamycins.

Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.

Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy

Visualization of DNA–protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3′-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA–protein interactions by AFM.

Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture ofa minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.