Dynamical Mechanisms For The Operation Of Transcription Apparatus

Since the proposal of the central dogma of molecular biology at the end of1950s, the molecular basis of transcription has become a fundamental subject in life science. Till the beginning of this century, the structure of the molecular machine orchestrating transcription, i.e., the transcription apparatus (TA), has been largely determined. However, it keeps unknown how the TA dynamically operates. The function of TA is to detect the time-varying cellular signals and initiate mRNA transcripts with appropriate rates. Traditional models stated that the response of TA to the variation in the concentration of transcriptional activators is almost continuous. However, it was observed that mRNAs are produced in a burst-like way. How the TA operates is still beyond the detection of available technologies till now.The main results of this work are:1) The dynamic principles of how the TA operates are presented, with related phenomena and conflicting concepts explained and unified;2) The contradictory viewpoints on the "transcription clock" are reconciled, with the essential dynamic mechanisms recovered;3) How the TA operates on a eukaryote-like prokaryotic promoter is revealed, providing a clue to the evolution of TA operation. The details are as follows.The TA in eukaryotes is a huge molecular machine. Based on the general structural organizations of the TA, this study proposes how the TA dynamically orchestrates transcriptional responses. The activators rapidly cycle in and out of a clamp-like space temporarily formed between the enhancer and the Mediator, with the concentration of activators encoded as their temporal occupancy rate (RTOR) within the space. The entry of activators into this space induces allostery in the Mediator, resulting in a facilitated circumstance for transcriptional reinitiation. The reinitiation rate is much larger than the cycling rate of activators, thereby RTOR guiding the amount of transcripts. Stochastic simulations based on this mechanism qualitatively reproduce and interpret up to7different profiles of gene expression, e.g., transcriptional bursting is not mere noise as traditionally believed, but rather the basis of reliable transcriptional responses.Recently, an interesting phenomenon called "transcription clock" has attracted wide attention:during gene transcription, proteins appear to cycle on and off some gene promoters with both long (tens of minutes) and short periods (no more than several minutes). This study analytically describes the state evolution of promoters in terms of DNA-protein interactions, with the molecular interactions associated with macroscopic measurable quantities. Through theoretical derivation, the traditional contradictory viewpoints are reconciled. Results show that the fast cycling dictates how the proteins behave on the promoter and stable binding hardly occurs. Different kinds of proteins rapidly bind/unbind the promoter at distinct transcriptional stages performing their functions; this feature is essentially manifested as slow cycling when detected by ChIP assays. Thus, the slow cycling represents neither stable binding nor external modulation. This work also reveals the relationship between the essence and measurements of transcriptional dynamics.Although the transcription dynamics in eukaryotes are hard to measure, resent progresses revealed the kinetics of RNA polymerase holoenzyme interacting with a remote enhancer-regulated prokaryotic promoter. However, it is still a great challenge to reveal how the whole TA operates. Based on structural analysis, this study proposes a dynamic mechanism that allows for interpretation of relevant experimental data. This study reveals the dynamic mechanism which reconciles all experimental data:there exists a rather unstable DNA loop bridged by two DNA binding domains of an activator. This implies that weak interaction between proteins and DNA sequence can play no subsidiary functions in regulating transcription. This work also provides a clue for the evolution of the dynamic mechanism of transcriptional regulation.In sum, based on structural data and employing the methods of physics and mathematics, this thesis explores how the TA dynamically operates in both eukaryotes and eukaryote-like prokaryotes. Our studies reconcile contradictory viewpoints on transcriptional dynamics, interpret a wide variety of experimental observations, and provide novel insights into gene transcription.