NSF Award
9405618
Abstract 9405618 The goal of this project is to understand the mechanisms in which polymerases transcribe through nucleosomes. The overall hypothesis to be tested is that transcription-induced positive stress disrupts histone-histone interactions within the nucleosome such that the nucleosome unfolds. The individual histone-DNA interactions are subsequently disrupted by the polymerase one histone at a time. The disruption of histone-histone contacts is seen as a pre-requisite to efficient release of the individual histones. To test this hypothesis the transcriptional effect on nucleosomes is divided into two components, transcription-induced stresses which is an indirect effect and polymerase-DNA interactions which is a direct effect. Closed circular DNAs containing varying levels of positive stress are reconstituted with either histone H2A,H2B or H3,H4 and in combinations. The interactions are characterized by treatment with competitor DNAs and subsequent analysis on sucrose gradients and non-denaturating agarose gels. More detailed information regarding histone-DNA and histone-- histone interactions are obtainable by DNAase I foot-printing and protein crosslinking. The direct effect of RNA polymerase is studied by utilizing a well defined polymerase, T7 RNA polymerase, and constructing 145 bp DNA fragments which position the elongating polymerase at precise positions on those DNAs. By reconstitution of the polymerase-DNA complex with histones and analysis by hydroxyl radical footprinting, it will be possible to evaluate the disruptive influence of a polymerase on histone-DNA and histone-histone interactions. In vitro transcription experiments will be done to correlate the previous results with the authentic process. Previous studies have indicated that transcription-induced stresses can be eliminated by inclusion of RNAase A which removes the RNA as the polymerase transcribes. It is therefore possible to assay the transfer potential of the individual histones in the presence or absence of these stresses by inclusion of a competitor DNA. Selective transfer of specific histones may be expected dependent on whether the induced stresses are present. This variability in transfer is expected to influence transcriptional efficiency. It is expected that the results of these experiments will provide a further understanding of mechanisms that facilitate transcription and may also have broad application to an understanding of the histone-DNA dynamics which occur in the regulation of genes by sequence-specific transcription factors. %%% The DNA of a eukaryotic cell is packaged into semi-regular repeated structures that are called 'nucleosomes'. In each nucleosome, 145 bp of DNA are wrapped in a left-handed coil 1 .8 times around two each of four highly basic proteins. These proteins, histones H3,H4,H2A, and H2B, require high NaCI concentration to cause dissociation from the DNA. These very strong binding energies are in two interactive forms: interactions with the DNA and protein-protein interactions between the histones. When all eight histones are in association with each other in a nucleosome, the combined binding energies of each histone with DNA generates an extremely stable structure. Any transient release of histone from the DNA would be prevented from total dissociation due to the continued interaction with the other histones in the complex. During transcription, the RNA polymerase enzyme must access this highly protected DNA. Therefore, gene regulation to a large extent is defined by nuclear mechanisms which are designed to circumvent these large binding energies. Transcription, DNA replication and repair, as well as the sequence-specific binding of factors which control these processes are all dependent on these same mechanisms. By disrupting histone-histone interactions within the nucleosome, histones H2A,H2B and/or H3,H4 could be transiently released such that RNA polymerase would be able to transcribe on a histone deficient template. We propose th at the source of the energy which disrupts these histone-histone contacts is the formation of transcription-induced stresses in the DNA. This hypothesis is tested by studying histone interactions with DNAs that have been constructed with topological stresses that mimic the stresses that would be produced by RNA polymerase. A further analysis is done with an in vitro transcription protocol which is designed to minimize variables. It is expected that the results of these experiments will provide a further understanding of mechanisms that facilitate transcription and will also have broad application to an understanding of the histone-DNA dynamics of a broad range of nuclear functions . ***
