The energetics of consensus promoter opening by T7 RNA polymerase

The 50-residue major coat protein (MCP) of Ff bacteriophage exists as a single-spanning membrane protein in the Escherichia coli host inner membrane prior to assembly into lipid-free virions. Here, the molecular bases for the specificity and stoichiometry that govern the protein-protein interactions of MCP in the host membrane are investigated in detergent micelles. To address these structural issues, as well as to circumvent viability requirements in mutants of the intact protein, peptides corresponding to the effective alpha-helical TM segment of wild-type and mutant bacteriophage MCPs were synthesized. Fluorescence resonance energy transfer (FRET) experiments on the dansyl and dabcyl-labeled MCP TM domain peptides in detergent micelles demonstrated that the peptides specifically associate into non-covalent homodimers, as postulated for the biologically relevant membrane-embedded MCP oligomer. MCP peptides labeled with short-range pyrene fluorophores at the N terminus displayed excimer fluorescence consistent with homodimerization occurring in a parallel fashion. Variant peptides synthesized with single substitutions at helix-interactive positions displayed a wide range of dimer/monomer ratios on SDS-PAGE gels, which are interpreted in terms of steric volume, presence or absence of beta-branching, and the effect of polar substituents. The overall results indicate discrete roles for helix-helix interfacial residues as packing recognition elements in the membrane-inserted state, and suggest a possible correlation between phage viability and efficacy of MCP TM-TM interactions.     

Initiation of RNA molecules by purified Escherichia coli RNA polymerase

Summary The kinesties of appearance of, and the distribution among, the four bases of the chain-initial nucleotides (5-termini) of RNA chains synthesized in vitro under various conditions have been investigated. The results of this study have shown that when native T4 bacteriophage DNA is the template for the RNA polymerase, most chains start with a purine nucleotide. The ratio of ATP termini to GTP termini is independent of the reaction time and of the template/enzyme ratio in the reaction mixture. A similar preferential purine initiation was observed when denatured T4 is the template, but the ratio of ATP to GTP termini is reduced. All 5-termini of poly-AU chains synthesized on poly-dAT templates are ATP.The determination of the kinetics of initiation of RNA chains has allowed direct confirmation of some conclusions which had been inferred previously from sedimentation analyses of the RNA product. (1) Most RNA chains are initiated during a short period at the outset of the reaction. (2) Low concentrations of native DNA templates limit the number of RNA chains synthesized, not the rate of RNA chain growth. (3) The maximum number of RNA molecules which can be synthesized on denatured DNA templates is severalfold larger than the maximum number which can be synthesized on the same weight of native DNA.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.     

Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase.

Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.     

Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase

E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.