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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Reorganisation of an RNA polymerase-promoter DNA complex for DNA melting.

Sigma factors, the key regulatory components of the bacterial RNA polymerase (RNAP), direct promoter DNA binding and DNA melting. The sigma(54)-RNAP forms promoter complexes in which DNA melting is only triggered by an activator and ATP hydrolysis-driven reorganisation of an initial sigma(54)-RNAP-promoter complex. We report that an initial bacterial RNAP-DNA complex can be reorganised by an activator to form an intermediate transcription initiation complex where full DNA melting has not yet occurred.

Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain.

We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP. We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2.

Communication between Esigma(54) , promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF sigma-dependent activator during transcription activation.

Conversion of Esigma(54) closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Esigma(54) activators is used in an energy coupling reaction to remodel the Esigma(54) closed promoter complex and to overcome the sigma(54)-imposed block on open complex formation. The remodelling target for the AAA activator within the Esigma(54) closed complex includes a complex interface contributed to by Region I of sigma(54), core RNA polymerase and a promoter DNA fork junction structure, comprising the Esigma(54) regulatory centre.

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.

Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element.

The sigma 54 promoter specificity factor is distinct from sigma 70-type factors. The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70.

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.

The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54.

Members of the protein family called ATPases associated with various cellular activities (AAA(+)) play a crucial role in transforming chemical energy into biological events. AAA(+) proteins are complex molecular machines and typically form ring-shaped oligomeric complexes that are crucial for ATPase activity and mechanism of action. The Escherichia coli transcription activator phage shock protein F (PspF) is an AAA(+) mechanochemical enzyme that functions to sense and relay the energy derived from nucleoside triphosphate hydrolysis to catalyze transcription by the sigma(54)-RNA polymerase.

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).

Mechanochemical ATPases and transcriptional activation.

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP.

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.

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.