8:00 PDT - 11:00 EDT - 15:00 UTC
16:00 BST - 17:00 CEST - 18:00 IDT
Department of Bioengineering, School of Engineering, University of California, Merced
Molecular mechanisms for gene tracking in eukaryotic transcription: Single-molecule analysis of the interplay between DNA recognition and folding of homeodomains
Transcription factors (TF) control gene expression by binding to their target DNA site to recruit, or block, the transcription machinery onto the promoter region of the gene of interest. Their function relies on the ability to find their target site quickly and selectively. The classical mechanism for such tracking is based on two DNA binding modes: high affinity specific to the target site and low-affinity non-specific (sequence independent). Non-specific binding competes for the TF’s occupancy by sheer numbers, but also enables the TF to slide along DNA resulting on facilitated diffusive search for the target along just one dimension. The TF can also transfer between DNA regions in transient spatial proximity as additional search-facilitating mechanism. These elements suffice to explain the homing, selectivity, and occupancy of prokaryotic TFs in living cells. In eukaryotes transcription control is much more complex, and operates in multiple layers, including dynamic control over the chromatin structure and epigenetic factors. However, even at the molecular level there are big unsolved puzzles. For instance, the DNA binding domains (DBD) of eukaryotic TFs recognize much shorter specific sequences (6-10 bp), but the genomes are orders of magnitude longer and hence contain thousands of randomly occurring, competing consensus sites. In addition, eukaryotic DBDs, and particularly homeodomains, are predicted to be intrinsically disordered based on their sequence and found to be flexible and highly dynamic in folding studies, which hints at a possible role for protein disorder in the DNA recognition process. Yet, these DBDs seem to form the same unique 3D structure when alone (by NMR) and bound to DNA (by X-ray crystallography).
In my laboratory we are addressing these molecular puzzles using single-molecule methods and the homeodomain from the eukaryotic TF Engrailed (EHD) as model system. Particularly, we are carefully mapping out the folding properties and DNA binding and diffusive behavior of EHD, both at the local level (<50 bp) and in the context of full genes using a combination of advanced sm-FRET methods, optical tweezers coupled to confocal fluorescence microscopy, and computer simulations. In this talk I will provide an overview of our prior work on the discovery of promiscuous DNA binding and transcription antennas, and on the identification of a disordered-enabled fast DNA gliding mode. I will also discuss some of our most recent results in support of a conformational rheostat mechanism for efficiently reading out the DNA sequence.