Richard R. Burgess, Ph.D. Professor Department of Oncology burgess@oncology.wisc.edu

Research Description:

Work in my laboratory focuses on Prokaryotic and eukaryotic RNA polymerases (RNAPs) and transcription factors and their roles in RNA synthesis and its regulation. We study the structure and function of the initiation factor, E. coli RNAP sigma70 subunit, by a concerted use of protein and physical chemistry, monoclonal antibodies (MAbs), molecular genetics, computer-based sequence and structure analysis, and biochemistry. We have overproduced and purified all seven known E. coli sigma factors and have made MAbs to them that can be used in measuring their levels in the cell under various growth conditions, inhibiting them, and immunoaffinity purifying them. We are carrying out systematic site-directed mutagenesis of sigma70 to determine more precisely the region involved in the binding of sigma to core RNAP (composed of alpha, beta and beta prime subunits). We have developed a powerful new method, using histidine-tagged RNAP subunit fragments, to map epitopes of our various MAbs easily and quickly. We have recently utilized a variation of this method, employing "far-Western blotting", to map interaction domains and have identified a major binding site for sigma70 within the region of amino acids 260-309 of the beta prime subunit of core RNAP. We will now be able to map the binding sites of a number of different proteins known to interact with core RNAP. We are also determining if the other E. coli sigmas bind to the same site on beta prime and where, precisely, the beta prime fragment binds to sigma70. Understanding these interactions in detail will allow us to better understand the complex but central mechanism of transcription and to design potentially important new antibiotics that work by disrupting transcription in pathogenic bacteria. We have developed a luminescence resonance energy transfer-based high-throughput screen to identify small molecules that interfere with sigma-core binding and might become lead compounds for drug development. We are using our unique "polyol-responsive" MAbs that are ideal for immunoaffinity purification of labile, multi-subunit complexes to rapidly isolate bacterial transcription machinery from crude extracts. Since some of our MAbs cross-react broadly among bacterial species, we are in an excellent position to collaborate with researchers trying to purify RNA polymerases from pathogenic bacteria.

We are utilizing special "polyol-responsive" MAbs that we have discovered to immunoaffinity purify RNAP II from human and yeast cells. We are focusing on the subunit architecture of yeast RNAP II and the interactions of the RNAP subunits with transcription factors and DNA. We have collaborated with R. Kornberg at Stanford to determine to crystal structure of yeast RNAP II. We are studying the interactions of human RNAP II with the basal transcription factors, TBP and TFIIB, at a minimal promoter. We are trying to determine which eukaryotic transcription factor(s) is the homolg of the prokaryotic sigma factors. We are also studying the role of specific transcription factors in normal and cancer cells and trying to develop single chain antibodies that will specifically interfere with its function in vitro and in vivo.

Docking model of bacterial RNA polymerase subunit b’260-330 coiled coil and s70 region 2.1-2.2

Predicted docking model generated by utilizing Hex docking software and structures from T. aquaticus core RNA polymerase and E. coli s70 (Current Opinion in Microbiology, 4:126-131, 2001). Specific side chains that, when mutated, abolish binding are indicated. The rudder is a feature adjacent to the b' coiled coil and is presented to enable orientation. This model represents a primary site of interaction between the sigma transcription factors and the core RNA polymerase discovered in our lab. Recently we have developed a luminescence resonance energy transfer-based high through-put screen based on this central cellular interaction and are using it to identify small molecule inhibitors that might be a new class of broad-spectrum antibiotics.

Selected Recent Publications: Articles on PubMed

• Burgess RR. (2007). Adaptation in bacterial flagellar and motility systems: from regulon members to 'foraging'-like behavior in E. coli. Nucleic Acids Res. 35:4441-4452. PMID 17576668
  
  
• Probasco MD, Thompson NE, and Burgess RR. (2007). Immunoaffinity purification and characterization of RNA polymerase from Shewanella oneidensis. Protein Expr Purif. 55:23-30. EPMID 17507238
  
  
• Sabree ZL, Bergendahl V, Liles MR, Burgess RR, Goodman RM, and Handelsman J. (2006). Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor. Gene. 376:144-151. PDF PMID 16698197
  
  
• Duellman SJ and Burgess RR. (2006). Overproduction in escherichia coli and purification of Epstein-Barr virus EBNA-1. Protein Expr Purif. 47:434-440. PDF PMID 16403648
  
  
• Thompson NE, Jensen DB, Lamberski JA, and Burgess RR. (2006). Purification of protein complexes by immunoaffinity chromatography: application to transcription machinery. Genet Eng (N Y). 27:81-100. PMID 16382873
  
  
• Lamberski JA, Thompson NE, and Burgess RR. (2006). Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody. Protein Expr Purif. 47:82-92. PDF PMID 16216525
  
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