Identifier

etd-04062005-152112

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. This study presents a biochemical characterization of a novel MarR homolog, HucR (hypothetical uricase regulator), from the DNA damage-resistant eubacterium, Deinococcus radiodurans. Circular dichroism spectroscopy suggests that HucR has ~47% alpha-helix and 10% beta-strand conformation at 25 deg C, and undergoes a transition to a disordered state with Tm = 51.1 ± 0.0 deg C. HucR binds as a homodimer with high sequence-specificity to a single site in its promoter region (hucO) with an apparent Kd = 0.29 ± 0.02 nM. DNaseI and hydroxyl-radical footprinting indicate HucR binding site sizes of ~24 bp and 21 bp, respectively. The binding site contains a pseudopalindromic sequence comprised of 8 bp inverted repeats separated by 2 bp that overlaps predicted promoter elements for hucR and a putative uricase (dr1160). Specific phenolic weak acids, notably uric acid, antagonize HucR-hucO complex formation. In vivo, uric acid increases transcript levels of hucR and dr1160, ~1.6-fold, and stimulates uricase activity 1.5-fold. HucR-hucO complex formation involves protein conformational changes and a decrease in the helical twist of the DNA duplex. Intrinsic fluorescence measurements show that uric acid induces HucR conformational changes, and its apparent Kd = 11.6 ± 3.7 micromolar and Hill coefficient of 0.7 ± 0.1 suggest negative cooperativity. An amino acid substitution in the predicted HucR wing (HucR-R118A) reduces DNA-binding affinity ~5-fold (Kd = 1.60 ± 0.14 nM), whereas a substitution in the predicted recognition helix (HucR-S104A) does not significantly alter DNA-binding affinity (Kd = 0.23 ± 0.03 nM). Each mutation decreases complex stability on the gel, but does not affect sequence-specificity. Intrinsic fluorescence spectra suggest altered conformations of the HucR-variants and altered mechanisms of DNA association. The mutations at HucR positions 118 and 104 also alter a predicted weak ligand-binding site, as indicated by minor changes in uric acid affinities for HucR-R118A and HucR-S104A (Kd = 9.7 ± 3.2 micromolar and 7.4 ± 0.5 micromolar, respectively) and modest attenuations of protein-hucO complex formation in response to uric acid.

Date

2005

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Anne Grove

DOI

10.31390/gradschool_dissertations.1748

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