RT Journal Article
SR Electronic
T1 RNA Synthesis is Associated with Multiple TBP-Chromatin Binding Events
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 045161
DO 10.1101/045161
A1 Hussain A. Zaidi
A1 David T. Auble
A1 Stefan Bekiranov
YR 2016
UL http://biorxiv.org/content/early/2016/03/22/045161.abstract
AB Cellular processes including transcription are inherently dynamic. Currently, the dynamics of transcription and other molecular processes in the cell are poorly understood because of a lack of methods that measure fundamental kinetic parameters in vivo. Precise estimation of the chromatin-binding on- and off-rates of general transcription factors (TFs) would allow stochastic modeling of pre-initiation complex formation2,3, RNA polymerase recruitment and elongation, and transcription4,5. Live-cell imaging at specific multi-copy genes is capable of yielding the residence time of TF-chromatin interactions at high temporal resolution (i.e., second timescale)6 but does not allow these measurements at single-copy genes. Cross-linking kinetic (CLK) analysis is a high spatial and temporal resolution method that enables estimation of the in-vivo TF-chromatin on- and off-rates at single-copy loci7. However, alternative approaches to estimating these kinetic parameters are needed to independently verify CLK as well as live-cell imaging approaches8. We developed and applied a physical modeling approach using chemical kinetic theory that directly estimates the residence time of TATA-binding protein (TBP) across the yeast genome from TBP competition ChIP data9 —another high-spatial resolution method10,11, which was generally believed to be low temporal resolution (20 min or greater). Using this approach, we are capable of estimating TBP-chromatin residence times on the minute time-scale across the yeast genome, demonstrating that competition ChIP is actually a relatively high temporal resolution method. Comparing TBP-chromatin residence times with nascent RNA transcription rates12, we find that ~5 TBP binding events are associated with productive RNA synthesis at the typical gene. Our results paint, for the first time, a highly dynamic, stochastic picture of pre-initiation complex formation with multiple rounds of partial assembly and disassembly before productive RNA polymerase elongation.