Mechanisms of Yeast TFIIB Function in RNAPII Transcription Initiation
Alfred Ponticelli Principal Investigator
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Ponticelli MCB-9905418<br/><br/>The regulation of RNA polymerase II (RNAPII) transcription plays a critical role in many complex eukaryotic processes. The long-term goal of this research is to understand the molecular mechanisms of RNAPII transcription initiation and the role of the general transcription factor IIB (TFIIB). Previous genetic and biochemical studies from this laboratory have identified several classes of TFIIB mutants in the yeast Saccharomyces cerevisiae that exhibit altered transcriptional properties in vivo and in vitro. In this project, some of these mutants will be utilized in combination with biochemical and molecular biological approaches to elucidate the mechanisms of TFIIB function in transcription initiation. In the first aim, the role of the TFIIB intramolecular interaction in the regulation of TFIIB activity will be determined. The association of TFIIB with promoter-bound TATA-binding protein (TBP) is a critical step in the formation of an RNAPII preinitiation complex (PIC). Previous studies identified a yeast TFIIB mutant (R64E) that exhibits increased activity in the formation of TBP-TFIIB-DNA (DB) ternary complexes and enhanced interaction between the N-terminal region and the C-terminal core domain. The parallel enhancement in DB complex formation and the intramolecular interaction in the R64E mutant suggests that the role of the intramolecular interaction in TFIIB is to promote the association of TFIIB with promoter-bound TBP. Gel mobility-shift assays and site-specific protein-protein photocrosslinking will be utilized to determine the role of the intramolecular interaction in regulating the entry of TFIIB into the transcription cycle. In addition, the solution structure of the yeast TFIIB N-terminal domain and/or the R64E protein will be determined in order to provide a structural basis to understand the functions of TFIIB in transcription initiation. In the second aim, the role of TFIIB in yeast transcription start site selection will be investigated. In contrast to mammalian cells, transcription initiation by S. cerevisiae RNAPII frequently occurs at multiple sites within a window of 45-120 base pairs downstream of the TATA element. The ability of yeast RNAPII to initiate within a window extending as far as 120 base pairs downstream of the TATA element reflects an interesting and fundamental difference in the initiation mechanism relative to that in mammalian cells. The basis for this difference is not known. A panel of yeast TFIIB mutants that confer downstream shifts in transcription initiation will be utilized in combination with DNase I footprinting, in vitro transcription assays with purified factors and site-specific protein-protein photocrosslinking to address two alternative hypotheses for the mechanism of yeast transcription start site selection and the role of TFIIB in this process, namely: 1) start site selection in yeast is directed by positioning of the PIC on the promoter and this positioning is determined by interactions involving TFIIB; and 2) start site selection in yeast involves a scanning polymerase, the properties of which are influenced by specific interactions involving TFIIB.<br/><br/>The first step in the expression of genetic information contained in DNA involves a process called transcription. The regulation of the transcription process plays a crucial role in governing the types of functions that a cell performs, the way in which a cell responds to environmental stimuli, and when and under what circumstances the cell will grow and divide. During transcription, messenger RNA is synthesized by the combined action of the enzyme RNA polymerase and a host of accessory proteins. An important goal towards a better understanding of the transcription process is to understand the functions and roles played by each of the accessory proteins. The work in this project is focused on determining the functions of the accessory factor TFIIB in transcription initiation.