This proposal will determine the structure of certain mammalian transcription complexes and describe their relationship to the control of both transcription and replication. First, in situ probing methods will be used to follow changes in SV40 nucleoprotein complexes as the viral lytic cycle proceeds. Altered viral templates, with specified functional defects, will be used to show which structures are important for which functions. The results will reveal the importance of the structural changes that accompany the replication-dependent changes in transcription patterns. Second, the communication between the SV40 origin of replication origin and the transcription elements will be investigated. Plasmids will be constructed that contain single and multiple copies of various transcription elements in the context of SV40 origin. Transcription and replication assays in cos cells will be used to learn how each process is affected by the presence of both types of elements. Cooperation of this kind is known to be important, and its mechanism will be further explored by following the changes in chromatin caused by the regulatory elements. Third, exploratory experiments will be conducted aimed at assessing structural changes that might accompany transcription initiation by polymerase II in vivo and in vitro. %%% A virus must transcribe certain of its genes in a defined manner to produce the enzymatic machinery necessary for viral development and replication. First, cells will be infected with the SV40 virus and at various times protein binding to specific regions of the viral genome will be determined. Once a region of the viral genome is found which is involved in protein binding, it will be altered in certain regions to assess the function of individual bases. The results will reveal the importance of sequence and structural changes that accompany the replication-dependent changes in transcription patterns. Second, the interactions between the SV40 origin of replication and the DNA sequences involved in gene expression will be investigated. Plasmids will be constructed that contain single and multiple copies of various transcription elements in the context of SV40 origin. Transcription and replication assays in cells will be used to learn how each process is affected by the presence of both types of structures. Interactions of these kind are known to be important, and their mechanism will be explored by following the changes in DNA-protein structure caused by the regulatory elements. Third, exploratory experiments will be conducted aimed at assessing any structural changes in the gene-protein complex that might accompany the initiation of mRNA synthesis.
