William B. Gurley

Professor

Undergraduate Coordinator  

Department of Microbiology and Cell Science University of Florida 
Ph. D. (1976) Department of Botany 
University of Georgia, Athens Georgia 
Postdoctoral: (1976-1979) Department of Plant Pathology 
University of Wisconsin, Madison, Wisconsin 

Teaching Interest

Plant and General Molecular  Biology
PCB 4522, Molecular Genetics (Spring)

Description of Research

General areas: Transcriptional regulation in plants and other eukaryotes; the heatshock  response, general transcription factors and upstream activator proteins;  the basic mechanisms involved in activated transcription.

Research in my laboratory is focused on two main topics of study: one  involving heat  shock transcription factors, and the other, transcription  factor IIB (TFIIB). All known organisms respond to a sudden elevation in  temperature (ca. 10°C) by turning off most gene expression and activating  a group of genes called "heat shock genes" that encode proteins that protect  the cell against the deleterious effects of high temperature. The protein  that regulates the induction of  heat shock genes is the heat shock transcription  factor (HSF). We have cloned 6 HSFs from soybean and 2 from Arabidopsis .Much to our surprise, only one of these transcription factors has been  able to stimulate transcription when assayed in eitheryeast or plant cells.We now believe that we have isolated a novel class of repressor HSFs thatensure that heat shock genes are not expressed under non-heatshock conditions  in plants. We are currently testing this hypothesisby expressing combinations  of active HSFs and repressor HSFs in transient assays involving the introduction  of the test genes (DNA) into plantcells by a variety of means including  particle bombardment and PEG-mediated transformation of protoplasts prepared  from leaf mesophyll cells.

Another objective of the heat shock research is to identify general  transcription factors of the preinitiation complex that serve as targets  of interaction for the HSFs. It is anticipated that identification of such  targets will provide insights into the basic mechanisms of activated transcription  in higher organisms and shed light on the process signal transduction,  from the perception of environmental stress to activation of heat shock  genes. We are conducting preliminary experiments using human HSFs since  much more is known regarding the process of transcription in animal cells. We have identified regions of  human HSF1 that make contact in vitro   with several general transcription factors including TFIIA (smallsubunit),  TFIIB, TAF55, TATA binding protein, and PC4 (a coactivator).This line  of investigation will also be pursued in plants.

The second research area involves characterization of general transcription factors in plants including TBP, plant TAFs, and transcription factor IIB (TFIIB). TFIIB is a key member of the preinitiation complex and servestoattach the RNA polymerase II/TFIIF complex to the TATA binding protein which, in turn, is bound to the promoter DNA. There are several points of difference between plant TFIIB proteins and those from animals and yeast.The next step is to determine whether these differences interfere with function when TFIIBs from plants are mixed in vitro with the transcription machinery from fungal and metazoan cells. In our continuing efforts to study the fundamental mechanisms of activated transcription, we have begun to clone and characterize TBP associated factors (TAFs) from Arabidopsis .   Antisense, T-DNA knockout, and RNAi approaches will be employedto deplete  specific TAFs and determine the effects on global transcription.

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PENDING PROPOSALS:

Characterization of TBP associated proteins (TAFs) in plants:
This project will assess the role of transcription factor TFIID and  TATAA binding protein (TBP)-associated factors (TAFIIs) in the transcriptional  expression of plant genes. The overall goal is to determine whether TFIID  is required for the expression of most genes transcribed by RNA polymerase II or is needed for only a minor subset of genes. This issue is being addressed  in animal and fungal systems, but almost no information is available regarding  higher plants. Major objectives: 1) Clone and characterize the major TAFIIs  from Arabidopsis. Function will be assessed by substitution of plant  TAFIIs in yeast and by monitoring activity of tethered-TAFII activity in  yeast and in plant cells using a double-site promoter. In addition, the  pattern of TAFII mRNA and protein expression will be determined in various  organs and at several developmental stages. 2) Evaluate the role of TFIID  in global transcription. The major TAFIIs will be depleted either through  RNAi (RNA interference) silencing of transformed Arabidopsis or using T-DNA  knockout  mutants. Transcriptional expression will be monitored by semi-quantitative  RT-PCR and RNA profiling using oligonucleotide chip-based microarrays.3) Obtain antibodies to the AtTAFIIs and TBP to determine TFIID composition by coimmunoprecipitation with TBP. These results will not only confirm  predictions regarding the identity of the cDNA clones, but immuno precipitation  of TFIID complexes in TAFII-depleted lines will identify key TAFIIs required  for TFIID stability.
The identification and characterization of coactivator proteins is vital to understanding the pathways of activated transcription in all higher  organisms. The cloning and characterization of Arabidopsis TAFIIsrepresents  a logical first step in the study of co-adaptor complexesin plants. This  information will not only provide added context tothe animal and yeast  studies, but will generally facilitate the development of strategies to  beneficially engineer plant gene expression. A detailed understanding of  plant gene activation should facilitate the design of specifically tailored  pathways for precise control of gene expressionand may contribute towards  the development of high expression systems for the synthesis of novel gene  products in plants such as peptidehormones or antibodies.

Arabidopsis TAFII225 as a ubiquitin domain protein:
The proposed experiments bring together two fields of study that heretofore have had little overlap: transcriptional regulation and protein degradation. Analysis of genomic sequences for Arabidopsis  TAFII225/205 indicate that these proteins contain an embedded ubiquitin  domain (UbD) and a ubiquitin activating/conjugating domain (ubac). The  experiments outlined here seek to clone the largest subunits of Arabidopsis  TFIID, TAFII225/205, and characterize the role of the embedded ubiquitin  domain. The main focus will be directed toward exploring the possible associationbetween TAFII225 and the proteasome under various conditions including  progression through the cell cycle, heat stress, simulated pathogen-attack,  and leaf senescence. Thesestudies will be conducted using Arabidopsis  suspension cultures,transformed plants, tobacco BY-2 cells, and in vitro ubiquitinationreactions. The possibility that TFIID may be targeted for  disassembly or degradation as a normal component of programmed cell death  is especially intriguing since senescence and cell death play such an active  rolein shaping the architecture of the plant throughout its vegetative and reproductive stages.
These studies are the first to explore the possibility that ubiquitin-mediated processes may regulate the stability or activity of a general transcription factor.The identification and characterization co-adaptor complexes, such as the  TAF subunits of  TFIID, is vital to understanding the pathways of activated  transcription in all higher organisms. The cloning and characterization  of Arabidopsis TAFII225 will not only provide added context to the  animal and yeast studies, but will generally facilitate the development  of strategies to beneficially engineer plant gene expression. A detailed  understanding of plant gene activation should facilitate the design of  specifically tailored pathways for precise control of gene expression and  may contribute towards the improvement of plant nutritional quality, or  the development of high expression systems for the synthesis of novel gene  products in plants such as peptide hormones or antibodies.

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Selected Publications

Czarnecka-Verner, E., Yuan, C.-X., Scharf, K.-D., Englich, K.-D. andGurley, W. B. (2000) Plants contain a novel multi-member class of heat  shock transcription factors without activator potential, Plant Mol.Biol. 43: 459-471.

Czarnecka-Verner, E., Pan, S., Yuan, C. -X., and Gurley, W.B. (2000).  Functional specialization of Plant Class A and B HSFs, In: Plant  Tolerance to Abiotic Stresses in Agriculture: Role of Genetic Engineering,  J. H. Cherry, ed., Kluewer Academic Publishers, Netherlands, pp. 3-28.

Yuan, C. -X. and Gurley, W.B. (2000). Potential targets for HSF1 within the preinitiation complex, Cell Stress & Chaperones 5: 229-242.

Pan, S., Czarnecka, E. and Gurley, W.B.  (1999).  Role o fthe TBP-TFIIB interaction in supporting basal and activated transcription  in plant cells, Plant Cell 12: 125-135.

Czarnecka, E. and Gurley, W.B. (1999). Plant heat shock transcription  factors:  divergence in structure and function, Biotechnologia 3:125-142.

Pan, S., Sehnke, P.C., Ferl, R.J. and Gurley, W.B. (1999).  Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex, Plant Cell 11:1591-1602.

Nagao, R. and Gurley, W. B. (1999) Use of  heat shock promoters  to control gene expression in plants, In: Inducible Gene Expression in Plants,  P. H. S. Reynolds (ed.), CAB International, pp. 97-126.

Czarnecka-Verner, E., Yuan, C. –X., Nover, L., Scharf, K. –D., English,G. and Gurley, W. B. (1998) Plant heat shock transcription factors: positiveand negative aspects of regulation. Acta Physiologiae Plantarum 19: 529-537.

Yuan, C. -X., Czarnecka-Verner, E. and Gurley, W. B. (1997). Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast. CellStress and Chaperones 2:263-275.

Baldwin, D. A. and Gurley, W. B. (1996) Isolation and Characterization of cDNAs encoding transcription factor IIB from Arabidopsis andsoybean. Plant J. 10: 561-568.

Czarnecka-Verner, E., Yuan, C. -X., Fox, P. C. and Gurley, W. B. (1995) Isolation and characterization of six heat shock transcription factor cDNA clones from soybean. Plant Mol. Biol. 29: 37-51.

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