Malcolm Buckle

FIS activates sequential steps during transcription initiation at a stable RNA promoter

FIS (factor for inversion stimulation) is a small dimeric DNA‐bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.

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 (t≈ 13 h) in the presence of Mn cations. Complexes were less stable when formed with Mg (t≈ 1 h). The role of Mn 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 (k ≈ 0.06 min) were obtained for the stable complex in the presence of either cation. Hence, the apparent preference observed for Mn in standard in vitro integration assays can be attributed entirely to the augmentation in the DNA binding affinity of the integrase.

DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen.

Singlet oxygen (1O2), generated by exciting an eosin‐Tris complex with a high intensity beam of radiation at 532 nm, was used to chemically modify bases in fragments of DNA containing the lac UV5 promoter in the presence of the DNA binding proteins, RNA polymerase and CRP (cAMP receptor protein). Subsequent treatment with piperidine selectively cleaved the DNA at specific modified bases in the sequence. Using this technique we show first that the reactivity of DNA bound by CRP differs in the presence and absence of RNA polymerase. Hence the local conformation of CRP‐bound DNA must change during the transition to the open complex. However, no reactivity is observed at the sites of the 40 degrees kinks described in the cocrystal structure (Steitz, 1990). Secondly we show that there is unique CRP‐dependent reactivity at a specific site (position −46 on the upper strand) in the open complex. Finally, in the open complex, 1O2 also reacts with sites 90 bp upstream from the transcription start point. This reactivity is qualitatively CRP‐independent. We infer that 1O2 reacts at sites where the promoter DNA is significantly distorted, and suggest that the pattern observed reflects the functional orientation of an active transcriptional complex in which the DNA is bent to form an extended loop.

Major histocompatibility class I molecules present Urtica dioica agglutinin, a superantigen of vegetal origin, to T lymphocytes

The Urtica dioica agglutinin (UDA) shares with the superantigens the property of activating T cell subsets bearing particular Vβ segments of the TCR. However, UDA is a lectin capable of binding to many glycoproteins on cell membranes. The implication of MHC versus other glycoproteins in UDA presentation was presently studied. Using mutant mice lacking MHC class I (MHC‐I), MHC class II (MHC‐II) or both MHC antigens, we provided evidence that MHC‐I and MHC‐II molecules serve as UDA receptors. Presentation by either one of these molecules ensured similar T cell responses and co‐stimulatory signals were mandatory for optimal T cell activation and proliferation both in MHC‐I and MHC‐II contexts. Remarkably, in the absence of MHC molecules, UDA could not be efficiently presented to T cells by other glycosylated proteins. Surface plasmon resonance studies were used to confirm the binding of UDA to MHC‐I molecules using a fusion protein consisting of MHC‐I domains and β2‐microglobulin. The results indicated that the interaction between UDA and MHC‐I molecules implicated lectin‐binding site(s) of UDA. Taken together, our data demonstrate that, in addition to MHC‐II antigens, MHC‐I molecules serve as an alternative ligand for UDA.

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. Copyright