Open Promoter Complex Research Articles

Visualizing RNA Extrusion and DNA Wrapping in Transcription Elongation Complexes of Bacterial and Eukaryotic RNA Polymerases

Transcription ternary complexes of Escherichia coli RNA polymerase and yeast RNA polymerase III have been analyzed by atomic force microscopy. Using the method of nucleotide omission and different DNA templates, E.coli RNAP has been stalled at position +24, +70 and +379 and RNAP III at position +377 from the starting site. Conformational analysis of E.coli RNAP elongation complexes reveals an average DNA compaction of 22nm and a DNA deformation compatible with ∼180° DNA wrapping against the enzyme. The extent of protein–DNA interaction attributed to wrapping, however, is less than that of corresponding open promoter complexes. DNA wrapping was also observed for RNAP III elongation complexes, which showed a DNA compaction of 30nm. When the RNA polymerases were stalled far from the promoter (+379 and +377), the growing RNA transcript was often visible and it was prevalently seen exiting from the enzyme on the opposite side relative to the smallest angle subtended by the upstream and downstream DNA arms. Surprisingly, we found that many complexes had a second RNAP, not involved in transcription, bound to the growing RNA of a ternary complex. DNA wrapping in the elongation complex suggests a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.

Enhancer-Dependent Transcription by Bacterial RNA Polymerase: The β Subunit Downstream Lobe Is Used by σ54 During Open Promoter Complex Formation

Publisher Summary This chapter describes experimental systems used in probing the function of the β subunit downstream lobe in the context of RNAP containing the major variant σ subunit, the enhancer-dependent σ factor, σ 54 . The protocols described in the chapter provide a simple step-by-step guide to obtain a relatively pure preparation of proteins required to study enhancer-dependent transcription. The experimental assays are designed to measure in vitro reconstitution of the σ 54 –RNAP and to assess the extent of DNA opening by the σ 54 –RNAP in response to activation. These assays are used in conjunction with potassium permanganate probing of wild-type and β(Δ186-433)E σ 54 –RNAP promoter complexes to show that RNAP β subunit residues 186–433 are used commonly by the enhancer-independent E σ 70 and enhancer-dependent E σ 54 for open promoter complex formation en route to transcription initiation.

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.

Two different modes of transcription repression of the Escherichia coli acetate operon by IclR.

IclR is a repressor for the Escherichia coli aceBAK operon, which encodes isocitrate lyase (aceB), malate synthase (aceA) and isocitrate dehydroge-nase kinase/phosphorylase (aceK) in the glyoxylate bypass. IclR also represses the expression of iclR in an autogenous manner. DNase I footprinting and in vitro transcription assays indicated that IclR binds to an IclR box (-21 to +14), which overlaps the iclR promoter and thus competes with the RNA polymerase for DNA binding, leading to transcription repression. In the case of the aceBAK operon, IclR binds to IclR box II between -52 and -19 of the aceB promoter and interferes with binding of the RNA polymerase to this promoter. A secondary IclR binding site (IclR box I) was identified between -125 and -99 of the aceB promoter. IclR binds to this IclR box I even after formation of the aceB promoter open complex and, moreover, induces disassembly of the open complex, leading to repression of aceB transcription. In parallel, the location of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) on DNA is shifted close to the IclR box I, indicating that direct interaction between the alphaCTD and the IclR box I-associated IclR caused the repression.

Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase.

To make messenger RNA transcripts, bacteriophage T7 RNA polymerase (T7 RNAP) undergoes a transition from an initiation phase, which only makes short RNA fragments, to a stable elongation phase. We have determined at 2.1 angstrom resolution the crystal structure of a T7 RNAP elongation complex with 30 base pairs of duplex DNA containing a "transcription bubble" interacting with a 17-nucleotide RNA transcript. The transition from an initiation to an elongation complex is accompanied by a major refolding of the amino-terminal 300 residues. This results in loss of the promoter binding site, facilitating promoter clearance, and creates a tunnel that surrounds the RNA transcript after it peels off a seven-base pair heteroduplex. Formation of the exit tunnel explains the enhanced processivity of the elongation complex. Downstream duplex DNA binds to the fingers domain, and its orientation relative to upstream DNA in the initiation complex implies an unwinding that could facilitate formation of the open promoter complex.

The Mechanism of Transcriptional Activation by the Topologically DNA-linked Sliding Clamp of Bacteriophage T4

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the σ-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer–template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation (∼320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 °C, but once opened at 30 °C remains open upon shift to the lower temperature. At a hybrid promoter for σ 70 and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

A Novel Bacteriophage-encoded RNA Polymerase Binding Protein Inhibits Transcription Initiation and Abolishes Transcription Termination by Host RNA Polymerase

Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli / X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, β′. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the σ subunit region 4 into the correct position for interaction with the −35 promoter element. As a result, open promoter complex formation on the −10/−35 class promoters is inhibited. Inhibition of promoter complex formation on the extended −10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination.

β Subunit Residues 186–433 and 436–445 are Commonly Used by Eσ 54 and Eσ 70 RNA Polymerase for Open Promoter Complex Formation

During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript. Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level. The Escherichia coli RNAP holoenzyme is a six-subunit (α 2ββ′ωσ; Eσ) protein complex. The σ subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the β subunit have profound effects on promoter melting by Eσ 70. The σ 54 subunit is a representative of an unrelated class of the σ subunits. Here, we determined whether mutations in the β subunit that affect late stages of promoter complex formation by Eσ 70 also influence promoter complex formation by the enhancer-dependent Eσ 54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Eσ 54 suggest that during promoter complex formation by Eσ 54 and Eσ 70 a common set of β subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Eσ 70 and Eσ 54 thus proceed through a common pathway.

Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex.

The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (alpha2betabeta'omegasigmaA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the sigma subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the sigma subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

Proteolytic DNA for mapping protein-DNA interactions.

We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, beta and beta', agree well with a recent model based on the crystal structure of the core enzyme alpha(2)betabeta' [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101, 601-611]. The cleavage site present on alpha supports previous studies regarding DNA binding regions of the alpha subunit. Cleavage sites identified throughout the sigma(70) subunit help to orient it with respect to the open promoter complex.

Interaction of RNA polymerase with forked DNA: evidence for two kinetically significant intermediates on the pathway to the final complex.

RNA polymerase forms competitor-resistant complexes with "forked DNA" templates that are double-stranded from the -35 promoter region through the first base pair of the -10 region, with an additional unpaired A at the 3' end of the nontemplate strand. These types of substrates were introduced recently as model templates for the study of DNA-protein interactions occurring in the early stages of the formation of RNA polymerase-promoter open complexes. We have performed kinetic and equilibrium measurements of interactions of wild-type and mutant RNA polymerases bearing substitutions in the sigma(70) initiation factor, with forked DNA of wild-type and mutant sequence. Our observations reveal that formation of a competitor-resistant complex between RNA polymerase and forked DNA, similar to the formation of open complexes at promoters, is a multistep process, and some of the sequentially formed intermediates along the two pathways share common properties. This work establishes, for the forked template, progression through these intermediates in the absence of downstream DNA and validates the use of forked DNA to determine the effects of changes in promoter or RNA polymerase sequence on the process of open complex formation.

Signaling of transcriptional chemistry in the on-line detection format

A critical analysis of optical and acoustic wave instrumentation for examining the transcription apparatus and its regulation is given in the present review. The physico-chemical parameters derived from such in vitro experiments are important from a biophysical standpoint. The overall mechanism of transcription is composed of several mechanisms such as DNA-binding and promoter selection, closed and open polymerase complex formation, initiation of RNA synthesis, elongation and termination. Surface plasmon resonance (SPR) and fluorescence spectroscopy are widely employed techniques for investigating these mechanisms in real time. Although the binding of nucleotides, transcription factors (TFs) and inhibitors to RNA polymerase (RNAP) and the DNA template have been studied extensively, the synthesis of mRNA has not been investigated in detail except by methods based on electrophoresis. The use of acoustic wave physics for investigating transcriptional chemistry offers not only a time-course analysis but also the potential to gain insight into structural changes that occur during the process.

The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening.

Evidence for post-recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutants of its Brf and B" subunits that are transcriptionally inactive because they fail to open the promoter. The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex. Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme. B" deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble. Removing an N-terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site.

Repression of Transcription Initiation at 434 P R by 434 Repressor: Effects on Transition of a Closed to an Open Promoter Complex

The lambdoid bacteriophage repressors function both as transcription activators and repressors. Regulation of transcription at the adjacent, but divergent promoters, P RM and P R, determines the phage's choice between the lytic and lysogenic development pathways. Here, we demonstrate that 434 repressor bound at 434 O R1 alone is not sufficient to repress transcription from 434 P R, but that 434 repressor bound at 434 O R2 alone is necessary and sufficient to repress P R transcription. This is different from what occurs in the related bacteriophage λ, in which binding of λ repressor to either λO R1 or λO R2 represses transcription from λP R. The combined results of gel mobility shift and KMnO 4 footprinting assays show that while 434 repressor binding to 434 O R2 does not preclude RNA polymerase binding at the P R promoter, it does prevent it from forming open complexes at this promoter. The RNA polymerase-P R complexes that form in the presence of repressor are heparin-resistant and the DNA is not melted. This observation indicates that 434 repressor bound at 434 O R2 inhibits transcription initiation at the P R promoter by “locking” the RNA polymerase-P R complex into an inactive state instead of “blocking” the access of RNA polymerase to promoter DNA.

Peculiar 2-Aminopurine Fluorescence Monitors the Dynamics of Open Complex Formation by Bacteriophage T7 RNA Polymerase

The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).

Dissection of Two Hallmarks of the Open Promoter Complex by Mutation in an RNA Polymerase Core Subunit

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.

The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates sigma-dependent transcription.

In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor sigmaH form of RNA polymerase (RNAP). The induction of sigmaH-dependent gene transcription requires the regulatory ATPase, ClpX. The ClpX-dependent post-exponential increase in sigmaH activity is not dependent on the activator of sporulation gene expression, Spo0A. By determining the level of sigmaH and sigmaA in whole-cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in sigmaA-RNAP holoenzyme. This is probably an indirect consequence of ClpX activity, because the ClpX-dependent decrease in sigmaA-RNAP concentration does not occur in a spo0A abrB mutant. The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but sigmaH-RNAP was observed to be more sensitive to ClpX-dependent stimulation than sigmaA-RNAP. No difference in transcriptional activity was observed in single-cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed- and open-promoter complex formation. ClpX is proposed to function indirectly in the displacement of sigmaA from core RNAP and to act directly in the stimulation of sigmaH-dependent transcription in sporulating B. subtilis cells.

Sequences within the DNA Cross-linking Patch of ς54Involved in Promoter Recognition, ς Isomerization, and Open Complex Formation

The bacterial RNA polymerase holoenzyme containing the final sigma(54) subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the final sigma(54) DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the final sigma and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by final sigma(54), and holoenzyme and is involved in activator-dependent final sigma isomerization. As part of the final sigma(54)-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the -12 promoter region to set basal and activated levels of transcription.

Conservation of sigma-core RNA polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes

Two distinct classes of RNA polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38). This indicates a common use of sets of sequences in core for interacting with the two sigma classes. Some sites chosen in sigma(54) for modification with Fe-BABE were positions, which when mutated, deregulate the sigma(54)-holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in sigma(54) may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of sigma(54) proximity to the core is apparently not evident with sigma(70), and may represent a specialized interaction.

Reiterative transcription initiation from galP2 promoter of Escherichia coli

The expression of gal operon in Escherichia coli is driven by two promoters, P1 and P2 separated by 5 bp. The transcription initiated from the P2 generates a large amount of abortive transcripts to produce a comparable amount of full-length transcript as P1 in vitro. In this study, we investigated the source of the abortive transcripts by employing a quantitative potassium permanganate footprinting method that determines the extent of open promoter complex formation. The extents of open promoter complex formation at the two gal promoters were about the same during the given reaction time while the amount of transcription initiation determined by in vitro transcription assay showed a considerable difference: several hundred-fold more transcription initiation from the P2 than the P1, most of which was abortive. Thus, it was concluded that the abortive transcripts are generated reiteratively by a small fraction of RNA polymerase. An in vitro transcription assay using an immobilized DNA template revealed that the fraction of RNA polymerase generating abortive transcripts never produces the full-length transcript and it remains bound to the promoter. We concluded that there are two kinds of RNA polymerase-promoter complexes formed at galP2, at least in vitro, productive complex and nonproductive complex; and, the nonproductive complex is responsible for generating large amount of abortive transcripts from the P2.