Abstract

Our structural and biochemical analysis of the RNA polymerase-factor(s) interactions will provide insights into how exactly protein-protein communications govern activities of different genes or sets of genes.

Issue

Signals from many cellular processes such as nutrient sensing, catabolite repression, hormone stimulation, differentiation, and responses to environmental stimuli ultimately reach the level of transcription to exert their effects. Aberration of transcriptional control is one of the mechanisms underlying tumoriogenesis. Oncogene products are frequently found to be key players in the control networks of signal transduction, cell cycle and transcription regulations. Using the model organism yeast, which shares extensive homology at molecular level with humans, we investigate the fundamental mechanisms of RNA polymerase II (Pol II). Pol II and its associated protein factors constitute the molecular machine that reads the genetic codes of chromosomes to synthesis mRNAs that serve as the templates to dictate protein synthesis. We use x-ray diffraction analysis to reveal the molecular structures of Pol II and its factors in space. Therefore, we can help understand the many aspects of the activities of these proteins (Pol II and its factors) involved in mRNA synthesis. Such molecular structural information will be extremely valuable to researchers working on rational drug design and therapeutic research. The outcome of this project will advance our understanding of how human gene-reading proteins function and how viruses such as HIV "hi-jack" the human system for viral propagation.

Response

We have developed and standardized a protocol for purifying large quantities of RNA polymerase II, a 12-subunit 514 kDa protein, from yeast. We have purified regulatory factors including Spt5 for forming co-crystals with the polymerase for structural analysis. We are now in a position to launch a full investigation of three distinct transcription complexes of RNA polymerase II using x-ray crystallography and biochemistry.

Impact

We have proved that large multiprotein-DNA complexes can be crystallized and therefore be analyzed by the powerful x-ray diffraction methods. This is the result of fours years of our effort with experiments in both biochemistry and x-ray crystallography. Through this, we have pushed the limits of structural biology and are in a position to generate new knowledge on the molecular mechanism that controls expression activities of genes.

Funding Sources

• Other Federal non-USDA (e.g., NSF, NIH, DOA, DOD)

Collaborators

• Memorial Sloan-Kettering Cancer Institute, New York, NY
• University of Maryland Schoold of Medicine, Baltimore, MD

Key Personnel

• Ping Ye, Cornell University
• Man Hee Suh, Cornell University
• Mincheng Zhang, Cornell University
• Peter Meyer, Cornell University
• Stewart Shuman, Sloan-Kettering Cancer Institute
• Christopher Lima, Sloan-Kettering Cancer Institute
• Averell Gnatt, University of Maryland School of Medicine, Dept. Pharmacology and Experimental Therapeutics