| Eukaryotic DNA is complexed with core histone octamers at ∼200 bp intervals. The histone octamer is a tripartite structure consisting of an H3/H4 tetramer flanked by two H2A/H2B dimers. Each histone protein has a structured domain and a highly conserved, basic N-terminal domain which is the site of several post-translational modifications (e.g., acetylation). Proteolytic removal of all the histone termini abolishes the condensed state, causing the array to exist mainly in the unfolded state. In this regard, my research focuses on elucidating the function(s) of the core histone N-termini in chromatin condensation. Previous studies suggest that folding and oligomerization are not strictly coupled and that different subsets of the histone tails (H2A/H2B or H3/H4) mediate different processes. To address this possibility, I have constructed hybrid trypsinized nucleosomal arrays. Sedimentation analysis has uncovered three distinct mechanisms by which the histone N-termini mediate folding and oligomerization.; Extensive folding of a nucleosomal array is repressive to transcription initiation and elongation, highlighting the importance of structure in gene regulation. Histone acetylation has been strongly correlated with the potential for transcriptional activity in vivo. To investigate how histone acetylation might potentiate active transcription; nucleosomal arrays of varying degree of acetylation have been assembled. Sedimentation and quantitative multi-gel electrophoreses analyses all indicate that histone acetylation change the hydrodynamic behavior of the arrays. Furthermore, in vitro transcription assays indicate that high levels of histone acetylation promote transcription under conditions that are normally repressive. Cumulatively, this indicates that histone acetylation. establishes the potential for active transcription by derepressing chromatin to an unfolded conformational state. These results yield new insight into the molecular basis of acetylation effects on both transcription and higher order compaction of nucleosomal arrays.; Transcription-related histone acetylation occurs in a very specific manner by type A histone acetyltransferases (HAT). Yeast Gcn5p, previously identified as a transcriptional adapter is a HAT-A enzyme. Initial characterizations of yeast rGcn5p reported an inability to acetylate nucleosomes. Given the dynamic nature of nucleosomal arrays in solution, the HAT activity of rGcn5p was reinvestigated. Under a narrow range of ionic conditions, both nucleosome core particles and nucleosomal arrays were acetylated by rGcn5p. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays ∼40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N-termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results indicate the structure and accessibility of the H3 N-termini changes upon assembly into nucleosomes. Moreover, the marked increase in acetylation. rates in folded nucleosomal arrays suggest that the location of the H3 N-termini are different in arrays compared to nucleosome core particles, supporting the idea of tail rearrangement during condensation.; The establishment of transcription-competent genes may occur during DNA replication. In this case, transcription factors would be utilizing H3/H4 tetramer sub-nucleosomal arrays. Both analytical hydrodynamic and electrophoretic techniques were employed to characterize H3/H4 tetramer arrays. Results indicate that the structural features of H3/H4 tetramer arrays closely resemble those of naked DNA. Furthermore, the cognate binding sites for transcription factor TFIIIA are significantly more accessible when the rDNA is complexed with H3/H4 tetramers than with histone octamers. These results suggest that the processes of DNA replication and transcription have evolved to exploit the unique structural properties of H3/H4 t... |
