Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.     

Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase

To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid approximately 9 bp upstream from the active site (at -9) it interacts with a region within the specificity loop (residues 744-750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172-191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination.     

Escherichia coli RNA polymerase binding to a DNA terminus prevents formation of a closed promoter complex

A 302 bp DNA fragment and a 113 bp subfragment of the former, both containing the fd gene VIII promoter (P VIII), were found to exhibit temperature-dependent differential behaviour in RNA chain initiation from P VIII. At 37 degrees C no significant differences were observed, while at 17 degrees C chain initiation was strongly suppressed only with the 113 bp fragment. This phenomenon depended on the presence of the (blunt) DNA terminus upstream from P VIII (position -70). Footprinting revealed that at 17 degrees C RNA polymerase was bound to this DNA fragment in a different mode. Contacts were observed only upstream from position -25. On the contrary, at 37 degrees C only the promoter complex footprint was visible. These results indicate that at 17 degrees C formation of the non-initiating complex is more favourable than formation of the promoter complex (which is closed at 17 degrees C; Hofer, B., Müller, D. and K?ster, H. (1985) Nucleic Acids Res. 13, 5995-6013) and that formation of both complexes is mutually exclusive. No footprints of RNA polymerase were observed at other DNA termini. This indicates a sequence-specificity for the interaction at the terminus of the 113 bp fragment. The footprint pattern, together with features of the DNA sequence, suggests that the contacts involved in this interaction are similar to those promoter contacts formed upstream from position -20 and that DNA without a -10 region can be specifically recognized by RNA polymerase.     

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.     

Regulation of RNA polymerase subunit synthesis in Escherichia coli: utilization of DNA-Intercalating drugs as a probe.

The effect of four DNA-intercalating drugs on the synthesis of the β and β′ subunits of Escherichia coli RNA polymerase was investigated. Acridine orange, proflavine, ethidium bromide, and berberine sulfate at sublethal doses caused a general reduction in cellular RNA and protein syntheses. Under this condition, acridine orange and proflavine rapidly led to overproduction of the β and β′ subunits in significant amounts. Ethidium bromide and berberine sulfate also caused overproduction of the two subunits but with a delay of 10 min at 30 °C. The β and β′ subunits of RNA polymerase became the major proteins being synthesized by E. coli cells after prolonged treatment with DNA-intercalating drugs. The level of the α subunit of RNA polymerase was not altered by any of the drugs tested. The overproduction of the β and β′ subunits induced by DNA-intercalating drugs is shown to require de novo synthesis of the ββ′ mRNA. These findings indicate that the expression of the ββ′ operon is regulated and that the synthesis of the α subunit is not regulated by the mechanism regulating the ββ′ operon. Taken together with evidence reported by others, these results strongly suggest that the concentration of intracellular free RNA polymerase plays a role in regulating the expression of the ββ′ operon.     

Structure of open promoter complexes with Escherichia coli RNA polymerase as revealed by the DNase I footprinting technique: compilation analysis.

Footprinting data for 33 open promoter complexes with Escherichia coli RNA polymerase, as well as 17 ternary complexes with different regulators, have been compiled using a computer program FUTPR. The typical and individual properties of their structural organization are analyzed. Promoters are subgrouped according to the extent of the polymerase contact area. A set of alternative sequence elements that could be responsible for RNA polymerase attachment in different promoter groups is suggested on the basis of their sequence homology near the hyperreactive sites. The model of alternative pathways used for promoter activation is discussed.     

A specific and efficient photoreaction between E. coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter.

Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine. The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex. It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template.