Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

Abstract

The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stoppedflow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8-to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/ RNAP-promoter rotational changes during 5-to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.abortive synthesis ͉ FRET ͉ rate-limiting ͉ T7 RNA polymerase ͉ transcription transition T he recruitment of RNA polymerase (RNAP) to the promoter and isomerization to a competent open complex are important regulatory steps in gene transcription (1, 2). Similarly, the transition from abortive initiation to processive elongation is an essential event of transcription that often dictates the rate at which the full-length RNA is made (3-9). During the abortive initiation phase, the RNAP maintains contacts with the promoter and catalyzes RNA polymerization by scrunching the template DNA (10-13). After a certain length of RNA is made, the RNAP switches transcription from promoter-specific to promoter-independent and processive RNA synthesis. Single-subunit and multisubunit RNAPs switch from the initiation complex (IC) to the elongation complex (EC) by undergoing specific structural rearrangements. For example, the single-subunit T7 RNAP protein undergoes major refolding of its N-terminal domain during EC transition that releases promoter contacts and results in the formation of RNA channel (14,15). The bacterial RNAP makes this transition by releasing the sigma factor from the promoter (7,(16)(17)(18)(19), and in eukaryotic RNAP II, this event is marked by the removal of several transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH (20). Although the initiation stage and the transition from IC to EC are major points of regulation that ultimately control gene expression, the rate-limiting steps governing these multistep processes and the kinetics of transition have not been elucidated.The challenge in studying the kinetic pathway of initiation and the IC-to-EC transition has been the transient and heterogeneous nature of the initially transcribing complexes. Studies of the transient heterogen...

New Insights into the Mechanism of Initial Transcription

“…The halt position-dependent profiles reveal that the wild type enzyme begins transitioning to elongation at about position ϩ8. This behavior is consistent with recent single molecule measurements that show a very slow transition to elongation commencing at position ϩ8, and increasing for complexes halted at positions ϩ9 and ϩ10 (20). Importantly, these data show that the P266L mutant is not transitioning to elongation significantly at position ϩ9, but instead the onset of its transition is delayed to about position ϩ11.…”

“…Fluorescence does not return to the fully free DNA base line. Halting at positions ϩ10 and beyond yields a more complete transition, indicating faster rates of transition to elongation, as seen previously (20).…”

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

“…Ha and coworkers showed that it is possible to perform FRET investigations with single-molecule sensitivity, [4] which has led to the wide spread use of single-pair FRET (spFRET) to investigate intra-and intermolecular distances and thereby conformations and dynamics in order to provide new insights into a number of biological questions. [5][6][7][8][9][10][11][12][13][14][15][16][17] SpFRET measurements can be performed either on immobilized molecules or on molecules in solution using a burst analysis. [18] In the solution-based experiments, the fluorescence of a dilute (of the order of 20-50 pm) solution of labeled molecules coming from the~fL volume of the focus of a confocal microscope is measured using a high numerical aperture objective.…”

The Histone Modifications Governing TFF1 Transcription Mediated by Estrogen Receptor

“…Intricate interactions between RNA polymerase II and its chromatin environment are essential for proper transcription progression through initiation, elongation, and termination (64,65). Although chromatin remodeling has been widely investigated in ER␣-regulated transcription, and various histone-modifying factors, including histone acetyltransferases p300/CBP and GCN5/pCAF (15,22) and histone methyltransferases CARM1, PRMT1, G9a, RIZ1, NSD1, MLL2, and SMYD3 have been implicated in this process, how histone modifications dictate ER␣-regulated transcription is still not fully understood.…”