ASSEMBLY OF POLYMERASE SPECIFIC TRANSCRIPTION COMPLEXES

Accurate transcription by each of the three eukaryotic nuclear RNA polymerases (pols) requires the assembly and recognition of unique transcription complexes on the promoters of appropriate genes. These complexes have at least one common component, the TATA box binding protein (TBP). In addition, complexes recognized by pols Il and III contain homologous proteins (TFIIB and TFIIIB70) that are known or believed, respectively, to interact directly with TBP. The properties of these molecules raise an important question: What mechanisms exist to ensure that a transcription complex with the correct polymerase-specificity will assemble on a particular gene? The long term goal of this proposal is to describe in quantitative terms, the biochemical basis for the assembly of RNA polymerase-specific transcription complexes. Initially, we propose to carry out quantitative analyses of the thermodynamics and kinetics of TBP-TFIIB and TBP-TFIIIB70 associations with model pol II and pol III promoters. Specially adapted enzymatic and chemical protection methods will be used for these studies. We will also analyze the assembly of TBP-TFIIIB70 and TBP-TFIIB complexes in solution by analytical ultracentrifugation. These experiments will be facilitated by using spectroscopically labeled TBP. Together, these studies will define quantitatively the chemical interactions that characterize the assembly of RNA polymerase-specific complexes. In the second phase of the work, spectroscopically-labeled transcription factors will be used to follow conformational changes of the TBP-TFIIB and TBP-TFIIIB70 complexes in solution and after binding to specific DNA templates. In the latter case, conformation changes to the DNA will also be followed using appropriate chemical and enzymatic probes. We will then correlate the conformational changes in the proteins and the DNA with specific kinetic or binding events resolved in the initial phase of the proposal. These data will provide knowledge of the mechanistic differences underlying the unique specificity of the complexes.