DNA structure variation at regulatory elements / Caryn M. Evilia.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Evilia, Caryn M.

Contributor:

Lu, Ponzy, 1942- advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Local Subjects:

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Physical Description:

xiii, 145 pages : illustrations (some color) ; 29 cm

Production:

1998.

Summary:

It is assumed that proteins induce structural changes in DNA. We hypothesize that regulatory DNA sequences assume an active role in protein interaction by adopting structures rendering them distinct from the surrounding DNA. We have studied DNA structure using multidimensional NMR and multiple isotopic labeling.

It has been previously observed that there is compression in the major groove at the start site of a T7 viral promoter, and a concomitant widening of the minor groove. To determine if this is also seen in more complex promoters, the bacterial lac and eukaryotic adenovirus major late promoters (AdMLP) were studied. Using 19F NMR, we observed similar results with the lac promoter. We also observed a widening major groove in the AdMLP TATA box corresponding to a bend in the published crystal structure. To obtain structural information on DNA, a protocol for synthesizing 13C/15N DNA was established and used to isotopically label a poly-A tract sequence that is thought to bend in solution. The spectra enabled us to generate a model of the solution structure for this sequence and demonstrate that this sequence is bending in the poly-A tract, in contrast to published crystallographic results.

The interaction of the tet repressor with its operator DNA was studied by 19F NMR. Published crystallographic data suggests that two amino acid residues in the DNA binding domain interact directly with the tet operator sequence, inducing a distortion in the DNA. Our results demonstrate that the tet operator displaces a base out of the helical axis in the absence of protein, and that the protein stabilizes this interaction. This suggests a novel interaction between the tet operator and repressor.

Additionally, a protocol for the synthesis of 5-fluoro-2' -deoxycytosine phosphoramidite was devised. This product was used to study an RNA tetraloop sequence that has been reported to form a hairpin structure by NMR and a double helix with an unusual C-U base pair by crystallography. The double-helical form was observed by a 19F-19F coupling under conditions identical to those used in the crystallographic experiments.

Notes:

Supervisor: Ponzy Lu.

Thesis (Ph.D. in Chemistry) -- University of Pennsylvania, 1998.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 99-13453.

OCLC:

187477658