DNA BINDING ACTIVITY OF RECOMBINANT TFII D PROTEINS

点击翻译按钮获取中文摘要

DESCRIPTION (Adapted from Applicant's Abstract): The regulation of gene expression is of central importance to the ability of the cell to respond to its environment, to control cell growth and to the process of development. Aberrant expression of some genes may lead to unregulated cell growth and the development of cancer. In most cases, gene expression is regulated at the level of mRNA synthesis. The applicant is studying the general transcription factors which are required by RNA polymerase II for the accurate initiation of mRNA synthesis at all promoter sites. The first of the general factors to bind to a DNA template in vitro is the TATA box transcription factor, TFIID. Binding of TFIID initiates the ordered assembly of the remaining components of the transcriptional machinery. Because of the difficulty in purifying the TFIID activity from mammalian and other metazoan cells, little is known about its biochemical properties. Recently, the gene encoding the yeast TFIID protein has been cloned and this allowed the isolation of cDNA clones of TFIID proteins from other species including human. Expression of the cloned TFIID proteins in bacteria provides sufficient quantities of active TFIID for detailed analyses of the biochemical properties of this important transcription factor. The studies proposed here will define the requirements for the formation of a functional TFIID protein-DNA complex. The equilibrium dissociation constant and the rate constants for binding and dissociation will be determined using a nitrocellulose filter binding assay and TFIID proteins purified from recombinant E. coli. The activation energy for DNA binding by wild type and an N-terminal deletion mutant of yeast TFIID will be measured in order to determine whether the N-terminus regulates TFIID-DNA binding. The sequence requirements for specific binding by TFIID will be examined using pools of oligonucleotide containing variant sequences in place of either a consensus TATA box element or in place of the sequences which normally flank a consensus TATA box element. Oligonucleotide which bind tightly to TFIID will be isolated following filtration through nitrocellulose and amplified by polymerase chain reaction. Repeated cycles of selective binding and amplification will identify the residues important for sequence specific binding by TFIID. Since the process of transcription is inherently an asymmetric process (the polymerase II molecule transcribes in only one direction from the start site of transcription) and since the binding of TFIID initiates the assembly of the transcription complex, it is possible that an asymmetrically oriented TFIID protein could nucleate the formation of an asymmetric complex. The symmetry of TFIID binding to a TATA box element will be determined by the analysis of the close contacts the protein makes with the DNA and by the use of a specific N-terminal peptide antibody.