The G+C-rich discriminator region of the tyrT promoter antagonises the formation of stable preinitiation complexes.

RNA polymerase forms a highly stable preinitiation complex at many prokaryotic promoters in the absence of ribonucleotides. These are often characterised by the longevity of the DNA strand-separated (open) complex in the presence of heparin. In contrast, such complexes are notoriously unstable at the promoters for rRNA and tRNA under similar conditions. The high G+C content within the DNA-melting region of these promoters has been implicated in this seemingly anomalous behaviour. Here, we used rapid-pulse UV laser photo-footprinting to monitor the transient structural intermediates formed at the Escherichia coli tyrT promoter. Promoter derivatives with A+T for G/C base substitutions within the G+C-rich discriminator region (-7 to -1) augmented the stability of complexes on both linear fragments and supercoiled plasmid DNA. Analysis of the lifetime of the preinitiation complexes as a function of the discriminator sequence reveals a direct relationship between the A+T content of the DNA-melting region and the stability of the ensuing complex. Our results are consistent with the premise that a G/C block to DNA-untwisting and/or DNA-melting operates to prevent the formation of the stable isomers that are implicated in most other transcription initiation pathways.

The kinetics of sigma subunit directed promoter recognition by E. coli RNA polymerase.

Time-resolved laser UV irradiation and controlled proteolysis have been used to study the sequential recognition of the lac UV5 promoter by Escherichia coli RNA polymerase. Local rearrangements in the DNA, the appearance of intimate protein-DNA contacts, and structural changes within the sigma subunit together provide specific signatures that define major species populated during this process. At 22 degreesC, a first closed complex is characterised by a transient conformational change in the sigma subunit and by a distortion in the -35 region. Subsequently, direct contacts at -34 and at positions -8, -5 and -3 on the non-template strand appear prior to DNA strand separation. The contact in the -35 consensus region involves only the sigma subunit. This intermediate possesses different structural parameters from that formed by quenching open complexes from 37 degreesC to 14 degreesC. Sigma thus appears as the principal partner acting during promoter recognition, a strongly coupled process involving two major intermediates only.

Real time in vitro analysis of transcription by RNA polymerase on immobilized DNA fibres.

We have used surface plasmon resonance (SPR) to follow variations in the concentrations of binary complexes as RNA polymerase moves into a transcriptionally competent initiation complex with immobilized DNA fibres containing promoter sequences. The use of SPR to follow complex binding phenomena is described. We have also followed the changes in the mass of initiation complexes following addition of the nucleotide triphosphates prerequisite for transcription on the immobilized template. These signals are interpreted in terms of the escape of RNA polymerase into elongation mode and the subsequent synthesis of nascent RNA molecules.

The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits a marked preference for Mn(II) rather than Mg(II).

In this investigation, we examine the interaction between the human immunodeficiency virus type I integrase and oligonucleotides that reflect the sequences of the extreme termini of the viral long terminal repeats (LTRs). The results of gel filtration and a detailed binding density analysis indicate that the integrase binds to the LTR as a high-order oligomer at a density equivalent to 10 +/- 0.8 integrase monomers per 21-base pair LTR. The corresponding binding isotherm displays a Hill coefficient of 2, suggesting that the binding mechanism involves the cooperative interaction between two oligomers. This interaction is quite stable, exhibiting a prolonged half-life (t1/2 approximately 13 h) in the presence of Mn2+ cations. Complexes were less stable when formed with Mg2+ (t1/2 approximately 1 h). The role of Mn2+ appears to be in the induction of the protein-protein interactions that stabilize the bound complexes. In terms of the 3'-end processing of the LTR, similar catalytic rates (kcat approximately 0.06 min-1) were obtained for the stable complex in the presence of either cation. Hence, the apparent preference observed for Mn2+ in standard in vitro integration assays can be attributed entirely to the augmentation in the DNA binding affinity of the integrase.

Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.

The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.