This research program focuses on the role of chromosomal proteins in the control of DNA conformation and accessibility as a template for RNA synthesis. Major emphasis is placed on the enzymatic mechanisms employed to modulate the interactions between DNA and DNA-binding proteins in chromatin. Of particular interest are those post-synthetic modifications of protein structure - such as acetylation, phosphorylation and methylation of histones, high-mobility-group (HMG) proteins and other nuclear proteins - that affect their charge and structure and thus influence their DNA-binding properties. The dynamics of these protein modification reactions offer the potential for rapid control of DNA accessibility to RNA (and DNA)-polymerases and other regulatory factors. A primary aim is to characterize the enzymes involved, the substrate specificities and the factors that control the extent of reactions that modify histones, HMG proteins and other components of chromatin. New affinity probes that permit the selective recovery of newly-phosphorylated or acetylated polypeptide chains have been developed to study the question of simultaneous modifications of the same polypeptide chain. New analytical methods are being developed for precise localization and quantitation of modified amino acids in histones and other nuclear proteins. The relationship between histone and HMG modifications, nucleosome structure, and the transcriptional competence of the associated DNA sequences is now being studied in systems that permit separation and comparisons of coding and non-coding regions of the same DNA strand (e.g. the coding and 'spacer' regions of ribosomal genes). A new approach to these problems is based on our recent finding that nucleosomes unfold during transcription to generate apparent half-nucleosomes with reactive -SH groups on histone H3 molecules. We propose to analyze the subunit structure of these unfolded nucleosomes, with particular attention to protein-protein and protein-DNA contacts, protein modifications, and associated enzyme activities. The reactivity of the histone H3 -SH groups is being exploited as a 'marker' for transcriptionally active nucleosomes and a device for their isolation. This has permitted the proof that histone H3 is hyperacetylated in the unfolded nucleosomes of genes transcribed by RNA polymerases I and II.
