Evidence for a Common Mode of Transcription Factor Interaction with Chromatin as Revealed by Improved Quantitative Fluorescence Recovery after Photobleaching

Abstract

How site-specific transcription factors scan the genome to locate their target sites is a fundamental question in gene regulation. The in vivo binding interactions of several different transcription factors with chromatin have been investigated recently using quantitative fluorescence recovery after photobleaching (FRAP). These analyses have yielded significantly different estimates of both the binding rates and the number of predicted binding states of the respective transcription factors. We show here that these discrepancies are not due to fundamental differences among the site-specific transcription factors, but rather arise from errors in FRAP modeling. The two principal errors are a neglect of diffusion's role and an oversimplified approximation of the photobleach profile. Accounting for these errors by developing a revised FRAP protocol eliminates most of the previous discrepancies in the binding estimates for the three different transcription factors analyzed here. The new estimates predict that for each of the three transcription factors, approximately 75% of the molecules are freely diffusing within the nucleus, whereas the remainder is bound with an average residence time of approximately 2.5 s to a single type of chromatin binding site. Such consistent predictions for three different molecules suggest that many site-specific transcription factors may exhibit similar in vivo interactions with native chromatin.

A single-molecule characterization of p53 search on DNA

“…It is also possible to estimate the minimal rate of the conformational transition in p53 required for fast search and specific binding. Using the measured diffusion coefficient for full-length p53 and in vivo time on DNA (22,23), we obtain a rate constant of about 10 3 s −1 (see SI Text). Thus, if the conformational transition happens on a submillisecond time scale, the protein can efficiently search for its cognate site.…”

“…Finally, we are able to relate our single-molecule measurements to published in vivo fluorescence recovery studies (22,23) and p53 copy-number measurements (25) to calculate the time it takes p53 to find a specific site (e.g., p21) on DNA (see SI Text).…”

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

“…Despite this fact, the diffusion constants were at least an order of magnitude smaller than those in our study. It is known, however, that when using FRAP it is very difficult to correctly account for restriction of the movement because of interactions (23). In addition, it is not straightforward to analyze mRNP mobility by FRAP if small fluorescent ligands are used to tag the particles, because the ongoing exchange of bound and free labels leads to complex recovery kinetics (24).…”

N- and C-terminal Domains Determine Differential Nucleosomal Binding Geometry and Affinity of Linker Histone Isotypes H10 and H1c

“…The role of the N-terminal domain is unknown and is proposed to be involved in proper positioning and anchoring of the globular domain onto chromatin. FRAP 2 is a powerful system for measuring protein dynamics in living cells (31,32). However, it does not account for biological and experimental variations leading to discrepancies in quantitative interexperimental conclusions and is prone to several drawbacks that can lead to faulty inferences (33).…”