The bridge helix is a 25 aa α helix spanning RNAP active site. It controls flow of nucleic acid substrates and nucleic precursors through catalytic site. Some models show that it is kinked, but others show it is straight. Periodic oscillation between straight and kinked was implicated in translocating RNAPs in single bp steps. Mutagenesis showed that mutations destabilising normal α helix conformation in certain positions increase specific RNAP activity. This increases frequency of Bridge Helix isomerisation between straight and kinked.
Bridge helix N-terminal contains a molecular hinge as well as C-terminal hinge region. When M808 was replaced with proline degree of superactivity exceeded highest level of superactivity in strongest C-terminal mutant.
Proline destabilises α helices due to lack of H bonding.
2 molecular hinges are referred to as BH-HN (Bridge Helix - Hinge N-terminal) and BH-HC (Bridge helix - hinge C-terminal). Proline substitutions increased BH kniking at 2 hinges, correlates with increased rate of nucleotide addition.
BH-HC kinking is stabilised by intramolecular interactions between aa side chains flanking each side of hinge. Local aa sequence predetermines location of BH-HC and its kinking properties.
BH-HN kinking involves G809 and G810 which are immediately C-terminal to M808 and invariant. Glycine show low helix-forming propensity as they are flexible. This enables glycines to flip out of α-helical conformation to create a flexible hinge. Flipped conformation is stabilised by noncovalent interactions of M808 with R811 and E812.
Mutagenesis shows any residue other than glycine reduces catalytic activity of RNAP.
A Recombinant RNA Polymerase II-like Enzyme capable of promoter-specific transcription by Werner and Weinzierl
They used a procedure to assemble active RNAP from recombinant subunits from archaeon Methanococcus jannaschii.
Expression, purification and in vitro assembly of recombinant M. jannaschii RNAP subunits
All subunits were expressed in recombinant form in E. coli. Subunits D-L and E-F were expressed as soluble heterodimeric xomplexes from a bacterila bicistronic system. Subunits H and P were expressed and purified under native conditions. A', A'', B', B'' and K were solubilised and purified by ion exchange chromatography in presence of 6M urea.
Subunits were assembled by combining qunatities of protines in buffer containing 6M urea. D-L-N-P subunits were added in at least 4M excess to drive assembly involving larger subunits to near completion.
Urea concentration was gradually lowered by stepwise dialysis against buffers containing decreasing urea concentrations. This favours renatiration and formation of scepfici protein-protein contacts.
Final dialysis steps in urea-free buffer under completely native conditions.
Structurally unstable and incomplete assemblies were removed by incubating renatured enzymes at 70°C for 10 min. This exploits intrinsic thermostability of correctly folded M. jannaschii polypeptides. Removes misfolded aggregates.
Recombinant archaeal RNAP is active in in vitro assays
Size exclusion chromatography shows that subunits coelute as a macromolecular complex. Presence of subunits was confirmed by Western blotting using antisera directed against these proteins.
To determine if assembly has catalytic properties, nonspecific transcription assay was carried out.
Promoter independent transcription activity was measured. α32P-rUTP was incorporated into material insoluble in 5% TCA in the presence of nicked ds template DNA. Peak of transcriptional activity (fractions 34-36) correponds to peak of correctly assembled RNAP. Presence of assembled RNAP is determined by size exclusion chromatography.
Purified TBP and TFB from M. jannaschii was shown to assemble on strong SSV T6 promoter by electrophoretic mobility shift assays.
To test if recombinant RNAP can interact with TBP/TFP/promoter complex, they added these components in in vitro transcription reactions. Primer extension assys detect formation of correctly initiated transcripts. Recombinant archael RNAP can carry out promoter specific transcription in a manner dependent on presence of BP and TFB.
A'-A''-B'-B''-D-L is closest approximation of archaeal RNAP to minimal bacterial core enzyme. Does not show measurable transcriptional activity. Unstable at elevated temperatures of heat treatment purification step.
rNTP incorporation into RNA transcripts only observed if supplemented with subunits N and P.
Contributions of subunits K, H, and E/F to catalytic activity
Recombinant K is insoluble under native conditions. It was incorporated into RNAP subunit assemblies using dialysis renaturation method. h and E-F are soluble and can be added to renatured enzyme complexes under native conditions.
To investigate K function. compare catalytic activities in presence or absence of K. Size exclusion chromotographyt shows K is efficiently incorporated into a soluble RNAP subunit complex.
Complexes lacking K in nonspecfici transcription assays shows it is no essential for catalytic activity. It enhances basal activity of minimal assembly A'-A''-B'-B''-DL-N-P 2fold.
To test H function, assemble RNAP complexes in presence of absence of H. Affects specfific activity of enzyme. H boosts activity by 10fold.
E and F form a stable heterodimeric complex. E-F is readily incorporated into recombinant RNAP. No detectable effects in promoter-specific or nonspecific transcription assays.
Active site mutagenesis supports functional roles for 2 Mg2+ bindings sites
Substituting absolutely conserved Asp with Ala in metal A motif destroys catalytic activity. Ala substitutions of 2 conserved carboxylates in metal B motif reduces specific activity. In E. Coli RNAP a mutation in metal B motif delays promoter clearance and slows elongation rate.
A Fully recombinant system for activator-dependent archaeal transcription by Ouhammouch
Ptr2 was shown to stimulate transcription at a very weak rb2 promoter. Subunits H,K, and E/F are not essential for basal r2 transcription.
Archaeal basal factor TFE is a homologue of 2 subunits of TFIIE in eukaryotes. TFE stimulates casal transcription at weak rb2 promoter.
Direct modulation of RNAP core functions by basal TFs by Werner and Weinzierl
Bridge helix and trigger loop perturbations generate superactive RNA polmerases by Tan and Wiesler
In many RNAP structures bridge helix is continuous and gently cruved α helix. In some bacterial RNAPs it is linker. Structural changes in trigger loop are thought to influence bridge helix conformations.
Residues that interact with rNTPs in catalyitc site are sensitive to mutagenesis. Substantial loss of function.
Some residues in spatially constrained positions can be replaced without substantial loss of function. Substituting A822 with residues containg large, bulky and/or hydrophobic side chains only decreases activity slightly. Tolerates proline substitution in certain BH positions.
Proline cannot participate in α-helix conformations, restricts conformational space of residue at its amino side and disrupts local H bonding that stabilises 2dary structure. In many positions of BH pro substitutions decreases activity greatly. In T821P and A822P increase of activity of mutant compared to wt. It is not required that any stage of nt addition cycle for BH to maintain continuous α-helical conformation.
Localised kinks in bridge helix cause superactive catalysis
Substitutions causing enhancement are in D816, Q817, Q823, S824 and M827 positions. Side chains of these residues point away from RNAP catalytic centre. Superactivity is not caused by mutated side chains stimulating active site, but due to conformational changes in BH or altered interaction pattern of BH with adjacent trigger loop.
Superactive S284P may cause kinked BH. When in α helix, Proline distorts helix by introducing kink.
The bridge helix coordinates movements of modules in RNAAP by Hein and Landick
Conformational flexibility is conferred by glycines at conserved locations. Weinzierl substituted proline at every position in helix. Most proline substitutions dramatically decreased RNA synthesis on nicked calf-thymus DNA. However substitutions at 808 and 824 adj to conserved glycines increased total RNA synthesis.
These positions correspond to locations of naturally occurring prolines in bridge helices of some bacterial RNAPs. Kinking at these 2 points seems to simulate RNA synthesis when facilitated by proline residue.
Bridge helix as a coordinator of conformational changes in RNAP
Weinzierl's fingins indicate that BH segments contact flexible loops in polymerase on either side of active site, downstream DNA channel and 2dary channel through which NTPs enter active site. These are amino- and carboxy-terminal hinges (HN and HC). HN and HC are adj to conserved glycines that may facilitate BH distottions and to regions that do not tolerate changes.
Hinge regions may facilitate helix distrotions for RNAP function.
|Structure of an elongation complex based on the crystal structure of a NTP-bound RNA polymerase from Thermus thermophilus (PDB 2o5j) . DNA (black) is melting into a transcription bubble that allows template-strand pairing with RNA (red) in a 9-10 base pair RNA-DNA hybrid. The bridge helix (cyan) and trigger loop/helices (yellow/orange) lie on the downstream side of the active site. The presumed path of NTP entry is indicated by the straight arrow. Interconversion of the trigger loop and trigger helices is indicated by the curved arrow. The RNA polymerase subunits are shown as semi-transparent surfaces with the identities of orthologous subunits in bacteria (α, β, and β', gray, blue, and pink, respectively), archaea (D, L, B, and A), and eukaryotic RNA polymerase II (RPB3, 11, RPB2, RPB1) indicated. The active site Mg2+ ions are shown as yellow spheres, and α,β-methylene-ATP in green and red. (b) Conformations of the bridge helix observed on crystal structures of a NTP-bound elongation complex and of an RNA polymerase lacking nucleic acids. The positions of nascent RNA, the template DNA strand, α,β-methylene-ATP, Mg2+, and straight bridge helix are from the PDB 2o5j structure. The looped-out bridge helix indicating the conformation in a nucleic-acid-free structure is from T. thermophilus RNA polymerase bound by σA initiation factor (PDB 1iw7). Positions at which substitutions with proline increase polymerase activity are marked by Cα spheres (HN and HC) . The location of a deletion of two amino acids in the plant RNA polymerase IV enzyme is marked by Cα-Cβ sticks (next to the white sphere marking the proline substitution). Sequences of the bridge helix from several RNA polymerases are shown, with the M. jannaschii bridge helix color-coded as in the molecular model: blue, segments in which two-amino-acid deletions eliminate polymerase activity; gray, segment in which deletions partially affect activity; white, segment in which deletions have minimal effect on activity; cyan, amino- and carboxy-terminal segments. Naturally occurring prolines at HN and HC are shown white-on-black.|
HN-proximal BH segment contacts 4 conserved loops in RNAP that form a cap to helix. That makes critical contacts with trigger helives, downstream fork junction of duplex and melted DNA, NTP substrate and nascemt RNA.
HC-proximal BH segment contacts clamp and switch regions 1 and 5 in an anchor. It changes conformation when clamp changes position or on formation of trigger helices. When trigger helices form, BH contacts to cap are reduced. This is consisistent with movement of central porition of helix towards trigger helices.
Increasing flexibility facilitates BH movements, explaining superactivity of proline substitutions at HN and HC.
Bridge helix conformation influences formation of trigger helices in response to DNA and RNA sequence or transcription regulators that interact with RNAP clamp, cap or anchor, affecting BH conformation through HN and HC.
A bridge to transcription by RNAP by Kaplan and Kornberg
The trigger loop is adjacent to the BH. It is conformationally flexible. It has been observed to interact with template-specified nt substrates. Mutations in trigger loop residues alter elongation rate, transcriptional pausin,g reponse to regulators, substrate selection and trasnscriptional fidelity.
Loop contacts BH.
Gain of function mutants: bridge and trigger may work together
Superactivating substitutions were identified in BH and trigger loop. Double-substitution mutants with gain of funtion subtitutions in both BH and trigger loop have not greater gain in activity that most severe single substitution. This indicates 2 domains function together to promote transcription.