HISTONES AND NUCLEOSOMES IN ARCHAEA (ARCHAEBACTERIA)

Ohio State University, Columbus OH

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Abstract

We have discovered that the Archaea (formerly Archaebacteria) contain small DNA binding proteins that have primary sequences, and predicted secondary structures, in common with the nucleosome core histones. These archaeal histones bind and compact DNA in vivo and in vitro into structures which visibly resemble nucleosomes. This discovery, coupled with the recent recognition that all the basic molecular components of transcription initiation are conserved in the Archaea and Eukarya (formerly Eukaryotes) suggests that the structure, and the functions of nucleosomes in genome compaction and in regulating gene expression, have evolved from a simpler system that is still retained in the Archaea. The experiments proposed are to determine if this is correct. We will determine the 3D structure of HMfA and HMfB, histones A and B from Methanothermus fervidus, and the molecular stoichiometry and architecture of the nucleosome-like structures (NLS) that they form. The protein-DNA interactions that determine where and how these archaeal histones bind and constrain DNA molecules into NLS will be determined by site-directed mutagenesis and by the isolation, selection and characterization of preferred and high affinity DNA binding sites. Nucleosomes are specifically positioned and re-positioned within chromatin to regulate eukaryotic gene expression at the level of transcription initiation, and we will determine if this is also the case for the archaeal NLS. We have established conditions that turn on and off four very strong archaeal promoters in vivo, and also procedures to isolate NLS assembled and cross- linked in situ in these cells under these different conditions. We will determine where NLS are assembled in vivo, in relationship to the sites at which these promoters direct transcription initiation, under conditions of promoter activity and inactivity, by identifying nuclease protected sites by probe hybridizations, indirect end-labeling and primer extension procedures. Determining how gene expression signals are accessed and activated from within chromatin remains one of the most important questions in biomedical research, and one of the most difficult to address experimentally. The experiments proposed will not only establish if histones, nucleosomes and chromatin originated in the Archaea, but also the extent to which this simpler system provides a valid model for studies of the structure of the nucleosome, of nucleosome positioning, and of the mechanisms by which nucleosomes regulate specific gene expression.