Mechanism and Regulation of Eukaryotic Transcription

Summary: Our laboratory studies the mechanism and regulation of the eukaryotic transcription machinery. Our long term goals are to understand (i) the mechanism of the general transcription factors in promoting transcription by RNA polymerase and (ii) the mechanism of transcription activation by gene-specific regulatory factors.

The research focus of the Hahn Laboratory is the mechanism and regulation of transcription. In eukaryotes, RNA polymerases are components of large protein machines that function to correctly position the polymerase at gene regulatory regions and integrate signals that switch transcription on and off. Most subunits of the transcription machinery are essential for viability and regulation of transcription is one of the key steps controlling cell growth, differentiation, and development.  Research in the laboratory aims to decipher key protein-protein and protein-DNA interactions within the transcription machinery and regulatory factors that are fundamental to the regulation and mechanism of transcription.

 Two central unanswered questions in the transcription field are: how do the general factors and polymerase together act to regulate transcription and, how is the transcription machinery influenced by gene-specific regulators? Our laboratory uses a multidisciplinary approach to explore fundamental mechanisms of transcription and its regulation that includes biochemistry, structural biology, and molecular genetics.

Current projects include the following:

Structure of the RNA Polymerase II Preinitiation Complex and the Mechanism of Transcription Initiation

One of the first steps in transcription involves formation of a large complex at gene promoters termed the preinitiation complex (PIC).  The PIC contains over 50 polypeptides and contains promoter DNA,  the general transcription factors and RNA Polymerase. We have developed a system for mapping protein-protein interactions within the PIC that utilizes site-specific photocrosslinkers, protein cleavage reagents, and a systematic method for identification of crosslinking targets. We have mapped the location of many general transcription factors and promoter DNA with respect to RNA Polymerase II and have derived a working model for the structure of the PIC and the mechanism of transcription initiation.  We are now extending this work to study the mechanism of DNA strand separation and other conformational changes in the PIC that occur during initiation.

 The Mechanism of Transcription Activation

Transcription activators stimulate transcription by recruitment of coactivator complexes and the general transcription machinery to promoters. We are focusing on the mechanism of acidic transcription activators that function in all eukaryotic organisms to activate transcription.  We are using the yeast factor Gcn4 as a model acidic activator.  Gcn4 contains two tandem acidic activation domains and directly regulates over 70 genes in yeast.  We found that both Gcn4 activation domains contact three common factors that are subunits of 4 different coactivator complexes.  One of these factors, Gal11, is a subunit of the Mediator coactivator complex and has at least two acidic activator-binding domains that are redundant for function.  Thus either of the Gcn4 activation domains can contact either Gal11 activator-binding domain to stimulate transcription. 

 None of the Gcn4 activator-binding domains has sequence similarity and an interesting problem is how a single activator can contact three apparently non-related proteins.  To address these questions, we are collaborating with Rachel Klevit’s lab at the University of Washington to determine the Gcn4-Gal11 structure.  We have solved the NMR structure for one of the Gcn4-Gal11 complexes and are extending this structural work to examine whether Gcn4 interacts with other activator-binding domains using similar principals.  We are also extending this work to examine other activation domains binding to a variety of targets.

 Regulation of Transcription by Coactivators

Coactivator complexes stimulate transcription by direct interaction with the general transcription factors and/or by remodeling chromatin to allow access of transcription factors to gene regulatory regions.  We study the class of coactivators that directly interact with the general factors (e.g., Mediator and SAGA complexes).  We have recently shown that SAGA directly interacts with the TATA binding protein (TBP) and recruits it to promoter regions.  This interaction is regulated by covalent modification of SAGA and we are now investigating the mechanism of this regulation and the biological consequences of modulating TBP-SAGA binding.