This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Transcription is the first step and the key control point in the pathway of gene expression. Transcriptional regulation underlies development, oncogenesis, and other fundamental processes. In eukaryotes the enzyme RNA Polymerase II (pol) is responsible for transcription of messenger RNA making pol s regulation central to gene expression. Pol contains 12 subunits massing about 0.5 Mda, but despite its size and complexity pol cannot recognize and initiate transcription from a specific promoter. In order to transcribe from a specific promoter pol requires at least five general transcriptions factors (IID, IIB, IIE, IIF and IIH). In addition to the five general transcription factors, pol requires the action of a 1.5 MDa complex called mediator in order to respond to activators and repressors of transcription. The purpose of this proposal is to use solution scattering to study the various complexes involved in activated transcription up to and including the 60 polypeptide complex of pol, the 5 general transcription factors and mediator. Structure determination of these large assemblies presents formidable technical challenges. We are attempting to meet these challenges through a combination of x-ray crystallography, electron microscopy, and solution x-ray scattering. This experimental proposal constitutes our research effort using solution scattering, which we believe fills the technological gap between x-ray crystallography and cryoEM in terms of its strength on submega Dalton complexes and its ability to study them in solution. The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription; it will establish a structural basis for studies of transcriptional regulation; and it may indicate how structures of other heterogeneous multiprotein assemblies can be solved in the future by the combined structural approach.
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