This research program deals with transcription and transcriptional regulation in one prokaryotic and one eukaryotic system. T4: The focus is on the enzymology of T4 late gene regulation, using defined and specifically constructed T4 late transcription units, located in plasmids. We propose these principal research topics: 1) Analysis of the roles of T4-specific RNA polymerase-binding proteins in T4 late promoter selection, RNA chain initiation, elongation and termination, and discrimination against other promoters; 2) Analysis of T4 late promoter specificity and selectivity by in vitro mutagenesis and synthesis of synthetic promoters; 3) Analysis of the coupling between DNA replication and T4 late gene activity in vitro. S cerevisiae RNA polymerase III: The focus is on the mechanism of specific transcription, on the assembly of stable transcription complexes and on the process of RNA chain formation in a transcription unit with internal promoters. Transcription components, including transcription factors, will be purified. The interaction of the large DNA-binding transcription factor with the internal promoters of tRNA genes will be further analyzed. Quantitative analysis of equilibrium binding constants for factor-DNA complexes will be undertaken. Complexes of the transcription factor with different promoters will be examined by electron microscopy. In a second series of studies, the interactions of a second transcription factor with these large transcription factor-DNA complexes will be analyzed in terms of DNA binding site, nature of bound proteins, stability and shape. In a third series of experiments complete transcription complexes, containing RNA polymerase III, will be assembled; DNA binding site, shape and stability will again be analyzed. RNA chain elongation will be analyzed, particularly from the point of view of the interaction of proteins with the internal promoter. Some experiments on the regulation of synthesis of transcription factor proteins in yeast are proposed.
| Kassavetis, George A; Prakash, Prachee; Shim, Eunjung (2010) The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening. J Biol Chem 285:2695-706 |
| Nechaev, Sergei; Geiduschek, E Peter (2008) Dissection of the bacteriophage T4 late promoter complex. J Mol Biol 379:402-13 |
