Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of electrostatic interaction of Escherichia coli RNA polymerase with promoters of T4 phage DNA]

A comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken. The data obtained indicate that there are some particular elements in the patterns of electrostatic potential distribution of promoter DNA specific for promoter groups differing by their functional response to ADP-ribosylation of the alpha-subunit as well as to rpoB403- or rpoB409 mutationals of the beta-subunit of RNA-polymerase.     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm-1 shift from 1306 cm-1 in aqueous solution to 1314 cm-1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm-1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm-1; in water, a strong hydrogen-bonding solvent, 1306 cm-1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm-1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12.

Summary Radioactively labeled 4.5S, 6S, and 10S RNAs from Escherichia coli were hybridized to EcoRI fragments from the E. coli genome. Each of these molecules bound to more than one DNA fragment. C_ot curve analysis of the kinetics of the annealing of these RNAs to denatured E. coli DNA suggests that the DNA corresponding to each of these molecules is reiterated in the genome. These experiments also suggest that these reiterated sequences are non adjacent.     

A kinetic model for interaction of regulatory proteins and RNA polymerase with the control region of the lac operon of Escherichia coli.

The proposed model deals with kinetic aspects of the interaction of repressor, CRP and RNA polymerase with the control region of the lactose operon and is formulated as a system of linear differential equations. Several variants of the model are considered. They differ in the assumed mechanisms which limit expression of the operon (due to diffusion of the molecules of polymerase to the promoter and/or due to a specific interaction of polymerase and promoter) and in the existence or non-existence of an indirect interaction between the molecules of repressor and CRP, when they are bound to the control region. An analysis of the model provides a unified interpretation for several phenomena connected with regulation of the lactose operon, in particular, for the dependence of expression on concentrations of regulatory proteins and for different patterns of expression in vivo and in vitro for a class of promoter mutations.     

Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.

A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters

The formation of complexes of RNA polymerases from E. coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique. The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged. The mutation does not interfere with the affinity of the enzyme for both initiating substrates. The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors. The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.     

E. coli RNA polymerase interacts homologously with two different promoters

We present and review experiments that identify points of close approach of the RNA polymerase to two promoters, lac UV5 and T7 A3. We identify the contacts to the phosphates along the DNA backbone, to the N7s of guanines in the major groove and the N3s of adenines in the minor groove, and to the methyl groups of thymines. These contacts to the two promoters are strikingly homologous in space, as shown on three-dimensional models, and identify major regions of interactions lying on one side of the DNA molecule (at -35 and -16), as well as further areas extending through the Pribnow box. Both promoters are unwound similarly by the polymerase, across a region of about twelve bases extending from the middle of the Pribnow box to just beyond the RNA start site. We discuss the areas of interaction in the context of promoter homologies and promoter mutations. The disposition of the contacts in space suggests a model for the pathway along which the RNA polymerase binds to promoters.     

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.