Transcriptional modulator Leu3p of yeast: Modular architecture and function
Abstract
The Leu3 protein of Saccharomyces cerevisiae is a pleiotropic trans-regulator that can function both as activator and repressor of transcription. Leu3p is a large protein (886 residues) that binds to upstream promoter elements (UAS$\sb{\rm LEU}$) of at least six genes. Leu3p-UAS$\sb{\rm LEU}$ complex formation was studied by analyzing the properties of a recombinant peptide Leu3p(17-147) that contains the DNA binding motif of the protein (residues 37-67) which belongs to the family of Zn(II)$\sb2$ Cys$\sb6$ clusters. Leu3p(17-147) is a monomer in the absence of UAS$\sb{\rm LEU}$, but assumes dimeric configuration when the DNA is present. The dimer interacts symmetrically with two contact triplets separated by 6 base pairs and approaches the DNA in such a way that each subunit is positioned closer to the one DNA strand than the other. The binding of Leu3p(17-147) to UAS$\sb{\rm LEU}$ is strongly affected by the spacing between the contact triplets and the type of the triplet. Leu3p(17-147) has been shown to retain the repressor function of the Leu3p. Repression is largely lost with a mutant peptide that has increased affinity for the DNA, indicating that there is no direct correlation between strength of binding and repression. The repression and activation functions of Leu3p are localized in different regions of the protein. The activation function was shown to be contained in the 30 C-terminal residues and to be necessary and sufficient for strong transcriptional activation in mouse fibroblasts. A set of serial deletions has limited the number of amino acids required for activation to a central region of 17 residues in the activation domain. An abrupt change in the activity, observed with a mutant missing the C-terminal 12 residues of Leu3p argues for the presence of an individual domain within the activation region. Circular dichroism analysis of a synthetic peptide, consisting of the last 28 amino acids of Leu3p, indicated the tendency to assume $\alpha$-helical structure in the presence of trifluoroethanol. Finally, Leu3p was shown to interact with the yeast TATA-binding protein (TBP) and is potentially involved in the recruitment of yeast TFIIB in the process to form a productive complex in vitro.
Degree
Ph.D.
Advisors
Kohlhaw, Purdue University.
Subject Area
Biochemistry|Molecular biology|Biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.