SUNY at Albany
June 19-23, 2001
Deformable Elements in the Promoter DNA as a Basis for Adaptive Conformational Transitions
Conformational transitions in DNA are reasonable considered as a structural basis underlying stability and functional properties of biologically significant complexes. Fine structural features in the respective DNA regions may therefore create a specific media for interaction with a particular ligand. Thus, promoter DNA of E.coli besides canonical hexamers may be annotated by the high frequency in the presence and preference in disposition of TA (at ?57, -52, -47, -40, -34, -29, -18, -12, -7, -1), while distribution of TG shows maxims at ?7, ?5, -27, -15, -7 and ?1 (1). Both these dinucleotides have high propensity to adopt unstacked helix configuration (2, 3), while their preferred disposition is optimal to enable rotational setting of promoter DNA on the surface of RNA polymerase (1). However potential deformability of T(A/G)s not necessarily means their flexibility in the genetic environment of promoter DNAs and we tested this possibility for promoters T7D and T7A1 using a combination of structure-specific reagents (DNAse I, S1-nuclease and potassium permanganate).
As expected, under standard experimental conditions T(A/G), particularly phosphodiester bonds flanking them at the 5¢-end, exhibited high sensitivity to DNAse. This reactivity depends on the reaction conditions and in the absence of magnesium ions central phosphodiester bond together with 1 or 2 neighboring bonds become DNAse-resistant. At least some thymines of T7D forming nuclease-resistant bonds (?50 and ?32) appeared to be available for modification with potassium permanganate, while ?50/-49 bond was attacked by S1-nuclease even in the presence of bivalent cations. It means that some T(A/G) steps are essentially deformed, while their helix configuration is easily rearrangeble. To test further this flexibility we used a cationic surfactant tetradecil-3-metil-amonium bromide (C12TAB) as a counter-ion and DNA-binding ligand. At low surfactant/phosphate ratios, when association is driven by electrostatic interaction of this ligand with phosphate moiety, C12TAB stabilizes DNA double helix increasing reactivity to DNAse in the vicinity of all T(A/G) and decreasing reactivity to potassium permanganate at ?50 and -32. At higher ratios, when C12TAB forms micelle-like particles on the DNA surface, we observed decreased nuclease sensitivity at all T(A/G) steps and dramatically enhanced reactivity to potassium permanganate for all thymines in these regions. Therefore, T(A/G) are capable to support adaptive conformational transitions even in the complexes with relatively small ligand.
Upon transcription complex formation potassium permanganate testing revealed unpaired thymines in the region, expected for local DNA melting (-12/+1). In the case of T7A1 additional deformation was found for TA at ?57, i.e. within contact area with alpha-subunits (-40/?64). DNA-binding domain of alpha has been recently annotated as a duplicated helix-hairpin-helix module (4), having a propensity to induce DNA bending in the target sites. Helix deformation at ?57 may reflect this bending, while preferential presence of T(A/G) at ?57, -52, -47 and ?40 may be required to facilitate alphas interaction with their multiple binding sub-sites. Intrinsic flexibility of periodically distributed T(A/G) steps is therefore consistent with their functional implementation.
This work is supported by the Russian Foundation for Basic Research (grant 00-04-48132).
References and Footnotes
O. N. Ozoline (1), I. S. Masulis (1) and V. A. Buckin (2)
(1)Inst./Cell Biophysics RAS, Pushchino, Moscow Region 142290, Russia Ph: 7-0967-739140; F: 7-0967-790509; email: firstname.lastname@example.org (2)Dep./Chem., Univ. College Dublin, Belfield Dublin 4, Ireland