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Our laboratory conducts research on gene transcription in the budding yeast Saccharomyces cerevisiae. The three main areas of investigation are described briefly below:
Structure, Function and Mechanistic Studies on the Tetratricopeptide
Repeat-Containing Subunit of TFIIIC.
Transcription by RNA polymerase III (pol III) is of fundamental importance in all eukaryotes since its products participate in a variety of essential cellular processes including protein synthesis, RNA processing and protein transport. The long-term goal of this work is to obtain a detailed biochemical understanding of the limiting steps in pol III gene transcription. To this end, we are studying the increased transcription associated with “activating” dominant and recessive mutations in the PCF1 gene of Saccharomyces cerevisiae. PCF1 encodes the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and is responsible for recruiting the heterotrimeric initiation factor TFIIIB to the DNA upstream of the transcription start site. The recruitment of TFIIIB is achieved by direct protein-protein interactions between TFIIIC131 and the TFIIB-related factor, TFIIIB70. The dominant and the recessive PCF1 mutations are thought to facilitate different partially limiting steps in the assembly of TFIIIB by affecting distinct protein conformational changes in TFIIIC131 and/or TFIIIB70. Biochemical experiments using purified yeast factors and recombinant proteins and molecular genetic strategies are being used to (i) identify the steps affected and the mechanisms of “activation” by the dominant and recessive PCF1 mutations, (ii) define the interaction domains between TFIIIC131 and TFIIIB70 and (iii) assess the role of TPR structure/function relationships in TFIIIB complex assembly.
Quantitative Thermodynamic and Kinetic Studies on Transcription Complex
The overall aims of this work are (i) to describe the reactions involved in the assembly of the heterotrimeric TFIIIB-DNA complex in terms of their biochemical parameters (equilibrium association constants and kinetic rate constants) and (ii) to understand the biochemical basis of the extraordinary stability of the TFIIIB-DNA complex (i.e. its resistance to dissociation by molar salt concentrations and polyanions). These studies have primarily used quantitative DNase 1 footprinting methods to determine thermodynamic and kinetic constants associated with the formation of complexes between TATA box-containing templates, the TATA box binding protein (TBP) and a pol III-specific homolog of TFIIB known as TFIIIB70. Current and future studies will include the final protein component of TFIIIB (TFIIIB90) and will add pre-steady state experiments (rapid reaction times), fluorescence based methods and analytical ultracentrifugation to the repetoire of approaches.
Secretory Signaling Pathway Regulation of Ribosome and tRNA Synthesis.
In this project, we are using genetic and biochemical systems to identify the components and the targets of a recently discovered signaling pathway (Li et al., Mol. Cell. Biol. 20, 3843-3851, 2000) that is responsible for coordinating the synthesis of ribosomes and transfer RNAs with cell growth.
If you are interested in our work and would like to explore research opportunities in the lab, email me at email@example.com