UVM Theses and Dissertations
Format:
Print
Author:
Brescia, Peter Joseph
Dept./Program:
Biology
Year:
2007
Degree:
MS
Abstract:
TATA-Binding Protein (TBP) is a highly regulated basal transcription factor required by all three RNA polyrnerases for transcription initiation on eukaryotic promoters. TBP is part of larger complexes of proteins known as TFIID, TFIIIB and SL1 that recognize the TATA element of DNA promoter sequences. In vitro transcription assays have shown that a TATA element alone cannot define a promoter leaving directionality and polymerase specificity to be influenced by additional promoter sequences. Understanding how these sequences affect transcription is vital to understanding this critical mechanism of cellular homeostasis.
Previous experiments have established TBP Promoter-Binding Factor (TPBF), a homotetramer, as a novel transcriptional regulatory factor that acts as both a positive transactivator by binding to the TBP Promoter Element (TPE), and as a repressor by binding to a negative cis-element (nTPE). The TPBF amino acid sequence from Acanthamoeba has not been shown to have significant homology to any known protein to date. Therefore, any structural information from this novel protein would add to our general understanding of transcriptional regulation. Efforts were directed toward investigating the protein structure by crystallization of an N-terminal truncation mutant of the TPBF protein bound to TPE containing DNA.
Due to the nearly symmetrical structure of TBP, binding to the TATA box may be in one of two orientations allowing transcription bi-directionally. TPBF has been shown to specify TBP transcriptional directionality but the mechanism of action is unknown. First, to determine the in vitro TBP binding orientation at TATA sequences and the role of TPBF and TFIIB during transcription initiation, TBP mutants have been constructed to allow site directed fluorophore labeling to allow Fluorescent Resonance Energy Transfer (FRET) analysis to be employed. It is expected that the TBP binding orientation and transcription direction be defined by the presence of the regulatory factor TPBF and/or the general transcription factor (GTF) TFIIB.
Second, in order to correlate in vitro findings with in vivo analysis, Micrococcal nuclease-TBP fusion proteins have been constructed. The fusion proteins will be purified from a recombinant bacterial system for in vitro transcription assays as well as stably transfected into Acanthamoeba castellanii for in vivo analysis. The calcium (Ca) inducible nuclease activity allows determination of TBP binding orientation at endogenous and experimentally defined promoter sequences.
Previous experiments have established TBP Promoter-Binding Factor (TPBF), a homotetramer, as a novel transcriptional regulatory factor that acts as both a positive transactivator by binding to the TBP Promoter Element (TPE), and as a repressor by binding to a negative cis-element (nTPE). The TPBF amino acid sequence from Acanthamoeba has not been shown to have significant homology to any known protein to date. Therefore, any structural information from this novel protein would add to our general understanding of transcriptional regulation. Efforts were directed toward investigating the protein structure by crystallization of an N-terminal truncation mutant of the TPBF protein bound to TPE containing DNA.
Due to the nearly symmetrical structure of TBP, binding to the TATA box may be in one of two orientations allowing transcription bi-directionally. TPBF has been shown to specify TBP transcriptional directionality but the mechanism of action is unknown. First, to determine the in vitro TBP binding orientation at TATA sequences and the role of TPBF and TFIIB during transcription initiation, TBP mutants have been constructed to allow site directed fluorophore labeling to allow Fluorescent Resonance Energy Transfer (FRET) analysis to be employed. It is expected that the TBP binding orientation and transcription direction be defined by the presence of the regulatory factor TPBF and/or the general transcription factor (GTF) TFIIB.
Second, in order to correlate in vitro findings with in vivo analysis, Micrococcal nuclease-TBP fusion proteins have been constructed. The fusion proteins will be purified from a recombinant bacterial system for in vitro transcription assays as well as stably transfected into Acanthamoeba castellanii for in vivo analysis. The calcium (Ca) inducible nuclease activity allows determination of TBP binding orientation at endogenous and experimentally defined promoter sequences.