Abstracts: Albany 2007, The 15th Conversation

Abstract

The TATA binding protein is essential for transcription initiation by all three classes of RNA polymerase. It is a two-domain protein in which the sequence of the carboxy-terminal domain (CTD) is strongly conserved (~80% sequence identity among eukaryotes), while that of the N-terminal domain (NTD) is poorly conserved both in size and in sequence. Yeast NTD contains about 60 residues and is highly charged, while human NTD is about 160 residues in length, is relatively uncharged and in addition contains an uninterrupted stretch of 38 glutamine residues. There is increasing evidence that expansions of the glutamine residues in human TBP are associated with neurodegenerative diseases such as Spinocerebellar Ataxia 17, Huntington's, and Alzheimer's. TBP binds promoter DNAs as a monomer but self-associates in the absence of DNA. Self-association may regulate the protein's availability for regulatory functions. Although these amino-terminal domains are poorly conserved, evidence indicates that they both provide a similiar function in modulating self-association of full-length yeast and human TBP proteins. Here we present a structural and functional comparison of yeast NTD (yNTD) and human NTD (hNTD) of the TATA binding protein. The isolated yNTD and hNTD both present evidence of self association, by pull-down, cross-linking, centrifugation sedimentation equilibrium, and centrifugation velocity experiments. However, there is some variation in oligermerisation, the yeast NTD velocity experiment give values for monomer, dimer, and upwards in increments of two, i.e., tetramer, hexamer and octamer, while hNTD gives values for monomer and tetramer. Fluorescence quenching experiments following the measurement of three tyrosines in hNTD differ from experiments following the measurement of one tryptophan in yNTD. Iodide appears to be the most efficient quencher of yNTD and cesium chloride the most efficient quencher of hTBP. Other factors being studied are the capability of yNTD and hNTD binding to the core domain and the spectrum obtained by circular dichroism experiments. Cooperativity, Crosslinking, and Supercoiling in the Interaction of AGT with DNAThe mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O 6 -alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents.Here, we report properties of the interaction between AGT and short DNAs and between AGT and covalently closed circular plasmids. We show that while AGT sediments as a monomer in the absence of DNA, it binds with high cooperativity to both single-stranded and double-stranded DNAs. This result is surprising in view of the 1:1 binding mechanism found in crystalline AGT-DNA complexes.Crosslinking analysis allows identification of protein residues juxtaposed in the cooperative complex. The simplest models consistent with the data place the Abstracts Albany 2007 The 15th...