This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator.

Regulation of gene expression can go awry for many reasons and sometimes diseases such as cancer are the result. Therefore, it is essential to understand the fundamental mechanisms controlling gene regulation. Chromosomal proteins play an active and critical role in the regulation of gene expression and in chromatin condensation. A characteristic of chromosomal proteins is that they bind to many different DNA sequences with moderate to high affinity using both a globular domain and unstructured basic 'tails'. In fact, gene activity and cell cycle controlled chromatin condensation are mediated by modifications such as phosphorylation and acetylation that occur in the unstructured 'tail' regions of histones. Yet, surprisingly little is known about the functionally active structures of the basic motifs because they are unstructured in the absence of interacting factors, and because structural and functional studies on other DNA-binding proteins have been confined primarily to globular domains of proteins that bind DNA sequence-specifically.

The proposed research uses a novel 'model protein' system to study the structures and binding properties of chromosomal proteins that have basic motifs such as 'SPKK' and 'AK' found in histone H1 'tails'. Techniques from molecular biology, biochemistry, and biophysics, with which the P.I. has much expertise, will be used. The specific aims in this proposal focus on four main issues concerning the structure and functional properties of these motifs: 1) Mutagenesis of the unstructured 'tail' region of the Drosophila chromosomal protein HMG-D protein followed by quantitative DNA binding studies to determine which structural motifs are induced by DNA binding. 2) Determination of how modifications such as phosphorylation affect structural and binding properties the motifs using in vitro phosphorylation and DNA binding studies. 3 and 4) Understanding how binding of the globular domain and unstructured chromosomal domains of HMG-D affect DNA structure and interactions in nucleosomal DNA using biophysical studies.

The long term goals of this research are to learn how modifications such as phosphorylation and acetylation affect the structural, binding, and functional properties of motifs in chromosomal proteins that are important in gene regulation. This work will help clarify how chromosomal proteins function at the molecular level. This understanding together with the rules already established for sequence-specific DNA recognition will provide new insights into the design of proteins tailored for a specific function in DNA binding. The methods that we use in this work may also prove to be useful in understanding other protein- DNA and protein-protein interactions that also utilize unstructured components that interact to form functional complexes.

R55GM052100

1995-09-01

1998-08-31