Organization of an activator-bound RNA polymerase holoenzyme.

Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor sigma(54) remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with sigma(54) alone, and of RNAP-sigma(54) with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-sigma(54) closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)--a multi-subunit enzyme responsible for gene transcription--by a small ( approximately 7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the "transcription bubble" near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.

Construction and functional analyses of a comprehensive sigma54 site-directed mutant library using alanine-cysteine mutagenesis.

The sigma(54) factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. sigma(54) is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define sigma(54) residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of sigma(54) was constructed by alanine-cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)sigma(54) were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of sigma(54) were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased sigma(54) isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after sigma(54) isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the sigma(54)-core RNAP interface changes upon activation.

The role of the conserved phenylalanine in the sigma54-interacting GAFTGA motif of bacterial enhancer binding proteins.

sigma(54)-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) sigma(54) interactions. Importantly, we provide evidence for a close relationship between F85 and the -12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the Esigma(54) closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed.

Interplay between the beta' clamp and the beta' jaw domains during DNA opening by the bacterial RNA polymerase at sigma54-dependent promoters.

The bacterial RNA polymerase (RNAP) is a multi-subunit, structurally flexible, complex molecular machine, in which activities associated with DNA opening for transcription-competent open promoter complex (OC) formation reside in the catalytic beta and beta' subunits and the dissociable sigma subunit. OC formation is a multi-step process that involves several structurally conserved mobile modules of beta, beta', and sigma. Here, we present evidence that two flexible modules of beta', the beta' jaw and the beta' clamp and a conserved regulatory Region I domain of sigma(54), jointly contribute to the maintenance of stable DNA strand separation around the trancription start site in OCs formed at sigma(54)-dependent promoters. Clearly, regulated interplay between the mobile modules of the beta' and the sigma subunits of the RNAP appears to be necessary for stable OC formation.

Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA.

The bacterial sigma54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein. Using DNA binding and transcription assays the ability of the enhancer-dependent sigma54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed. Different DNA regions important for stabilising sigma54 holoenzyme-promoter interactions, destabilizing binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking sigma54 Region I were identified. It appears that homoduplex structures are required for early events in sigma54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from -5 to -1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of sigma54 was shown to be involved in interactions with the sequences in the -5 to -1 area.

Correlating protein footprinting with mutational analysis in the bacterial transcription factor sigma54 (sigmaN).

Protein footprints of the enhancer-dependent sigma54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in sigma54 of potential functional importance at the interface between sigma54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of these positions. Properties of the mutant sigma54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of sigma54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other within core interacting sequences of sigma54, failed to stably bind the activator in the presence of ADP-aluminium fluoride, an analogue of ATP in the transition state for hydrolysis. Overall, the data presented describe a collection sigma54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the view that the interface between sigma54 and core RNAP is functionally specialised.

Beta subunit residues 186-433 and 436-445 are commonly used by Esigma54 and Esigma70 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 (alpha(2)betabeta'omegasigma; Esigma) protein complex. The sigma subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the beta subunit have profound effects on promoter melting by Esigma70. The sigma54 subunit is a representative of an unrelated class of the sigma subunits. Here, we determined whether mutations in the beta subunit that affect late stages of promoter complex formation by Esigma70 also influence promoter complex formation by the enhancer-dependent Esigma54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Esigma54 suggest that during promoter complex formation by Esigma54 and Esigma70 a common set of beta subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Esigma70 and Esigma54 thus proceed through a common pathway.

Sigma54-dependent transcription activator phage shock protein F of Escherichia coli: a fragmentation approach to identify sequences that contribute to self-association.

Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the sigma54-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the sigma54-RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of sigma54-dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer-protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the sigma54-RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to sigma54-RNAP binding and transcription activation.

Inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged strip of amino acid residues in Gp2.

Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known. Here, the in vivo and in vitro properties of altered forms of Gp2 with amino acid substitutions in the NCS are described. While mutations in the NCS do not compromise the folding or the ability of Gp2 to bind to the RNAp ß' subunit, disruption of the NCS significantly attenuates Gp2 function in vivo and its ability to inhibit RNAp in vitro. Efficient inhibition of the RNAp by Gp2 also involves the amino terminal region 1 domain of the RNAp promoter specificity subunit σ(70), located in the vicinity of the primary Gp2 binding site in ß'. The results are discussed in the context of hypothetical molecular mechanisms of RNAp inhibition by Gp2.

Reprint of: inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged strip of amino acid residues in Gp2.

Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known. Here, the in vivo and in vitro properties of altered forms of Gp2 with amino acid substitutions in the NCS are described. While mutations in the NCS do not compromise the folding or the ability of Gp2 to bind to the RNAp ß' subunit, disruption of the NCS significantly attenuates Gp2 function in vivo and its ability to inhibit RNAp in vitro. Efficient inhibition of the RNAp by Gp2 also involves the amino terminal region 1 domain of the RNAp promoter specificity subunit σ(70), located in the vicinity of the primary Gp2 binding site in ß'. The results are discussed in the context of hypothetical molecular mechanisms of RNAp inhibition by Gp2.

Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting.

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Esigma54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status.

Protein-DNA interactions that govern AAA+ activator-dependent bacterial transcription initiation.

Transcriptional control at the promoter melting step is not yet well understood. In this study, a site-directed photo-cross-linking method was used to systematically analyse component protein-DNA interactions that govern promoter melting by the enhancer-dependent Escherichia coli RNA polymerase (RNAP) containing the sigma(54) promoter specificity factor (E sigma(54)) at a single base pair resolution in three functional states. The sigma(54)-factor imposes tight control upon the RNAP by creating a regulatory switch where promoter melting nucleates, approximately 12 bp upstream of the transcription start site. Promoter melting by E sigma(54) is only triggered upon remodelling of this regulatory switch by a specialised activator protein in an ATP-hydrolysing reaction. We demonstrate that prior to DNA melting, only the sigma(54)-factor directly interacts with the promoter in the regulatory switch within the initial closed E sigma(54)-promoter complex and one intermediate E sigma(54)-promoter complex. We establish that activator-induced conformational rearrangements in the regulatory switch are a prerequisite to allow the promoter to enter the catalytic cleft of the RNAP and hence establish the transcriptionally competent open complex, where full promoter melting occurs. These results significantly advance our current understanding of the structural transitions occurring at bacterial promoters, where regulation occurs at the DNA melting step.

A role for the conserved GAFTGA motif of AAA+ transcription activators in sensing promoter DNA conformation.

Transcription from sigma54-dependent bacterial promoters can be regarded as a second paradigm for bacterial gene transcription. The initial sigma54-RNA polymerase (RNAP).promoter complex, the closed complex, is transcriptionally silent. The transcriptionally proficient sigma54-RNAP.promoter complex, the open complex, is formed upon remodeling of the closed complex by actions of a specialized activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family in an ATP hydrolysis-dependent reaction. The integrity of a highly conserved signature motif in the AAA activator (known as the GAFTGA motif) is important for the remodeling activity of the AAA activator and for open complex formation. We now provide evidence that the invariant threo-nine residue of the GAFTGA motif plays a role in sensing the DNA downstream of the sigma54-RNAP-binding site and in coupling this information to sigma54-RNAP via the conserved regulatory Region I domain of sigma54 during open complex formation.

Sensor I threonine of the AAA+ ATPase transcriptional activator PspF is involved in coupling nucleotide triphosphate hydrolysis to the restructuring of sigma 54-RNA polymerase.

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).

Coupling nucleotide hydrolysis to transcription activation performance in a bacterial enhancer binding protein.

The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved 'DE' Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved 'DE' Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the sigma(54)-dependent transcription activation process. We established their role in the regulation of PspF self-association and in the relay of the ATPase activity to the remodelling of an RNA polymerase.promoter complex (Esigma(54).DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP.bEBP.Esigma(54) complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the sigma(54)-dependent transcription activation process. In addition, we show the importance of this glutamate residue in sigma(54).DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway.

Stable DNA opening within open promoter complexes is mediated by the RNA polymerase beta'-jaw domain.

DNA opening for transcription-competent open promoter complex (OC) formation by the bacterial RNA polymerase (RNAP) relies upon a complex network of interactions between the structurally conserved and flexible modules of the catalytic beta and beta'-subunits, RNAP-associated sigma-subunit, and the DNA. Here, we show that one such module, the beta'-jaw, functions to stabilize the OC. In OCs formed by the major sigma70-RNAP, the stabilizing role of the beta'-jaw is not restricted to any particular melted DNA segment. In contrast, in OCs formed by the major variant sigma54-RNAP, the beta'-jaw and a conserved sigma54 regulatory domain co-operate to stabilize the melted DNA segment immediately upstream of the transcription start site. Clearly, regulated communication between the mobile modules of the RNAP and the functional domain(s) of the sigma subunit is required for stable DNA opening.

Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription.

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the sigma54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma54 contact is required for the sigma54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of sigma54 and sigma54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP.AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between sigma54, sigma54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of sigma54 and sigma54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.

Multiple roles of the RNA polymerase beta subunit flap domain in sigma 54-dependent transcription.

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.