Wartell, Roger M.

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    DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry
    (Georgia Institute of Technology, 1988-12-23) Wartell, Roger M. ; Adhya, Sankar Lal
    Gal repressor dimer binds to two gal operator sites, O[subscript E] and O[subscript I], which are 16 bp long similar sequences with hyphenated dyed symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1,3 and 8, and the rotational 1′, 3 and 8′ of the symmetries. We have shown that Gal repressor binding to O[subscript E] or O[subscript I] DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and O[subscript E] and O[subscript I] DNA. The CD spectral change was not observed when the central 8,8′ C-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1′ G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8′ base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.
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    Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters
    (Georgia Institute of Technology, 1987-01-26) Plaskon, R. Richard ; Wartell, Roger M.
    The regions upstream from forty-three procaryotic promoters were examined for nucleotide distributions which have been associated with DNA curvature. The analysis procedure assigned a DNA curvature score based on the phasing of the 5' and 3' ends of An and Tn tracts, n greater than or equal to 3. The weighting scheme for the curvature score was based on recent studies which showed that tracts of An and Tn periodically phased with the helix repeat cause DNA curvature. Results show that promoters which have high transcription initiation rates in vivo tend to have high curvature scores in their upstream regions. Regions downstream from the transcription start-point do not have sequences correlated with DNA curvature. Four promoters which have been shown to have upstream activation regions have curvature scores above 1.5 in their -40 to -150 regions. The correlations observed lend support to the hypothesis that DNA curvature is associated with upstream activation of transcription
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    The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter
    (Georgia Institute of Technology, 1985-10-25) DeGrazia, Henry ; Abhiraman, Saraswathy ; Wartell, Roger M.
    The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.