Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics

Toyotaka Ishibashi, Manchuta Dangkulwanich, Yves Coello, Troy A. Lionberger, Lucyna Lubkowska, Alfred S. Ponticelli, Mikhail Kashlev, Carlos Bustamante
2014 Proceedings of the National Academy of Sciences of the United States of America  
Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the
more » ... somal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression. optical tweezers | Pol II | yeast | enzyme kinetics T ranscription regulation is the first step in the control of gene expression and it is a fundamental and highly coordinated process in all cells. RNA polymerase II (Pol II) is responsible for the synthesis of mRNAs, most snRNAs, and microRNAs in eukaryotic cells. Transcription elongation by Pol II is regulated by many elements such as the state of Pol II phosphorylation in the C-terminal repeat domain (CTD), the presence and stability of nucleosomes, the extent and stability of the nascent RNA structure formed behind Pol II, and several transcription factors. However, many of the molecular details underlying Pol II transcriptional regulation remain unknown. When Pol II synthesizes an RNA transcript, the enzyme translocates along the DNA template by thermally fluctuating between the pre-and the posttranslocated states; the binding of NTP to the posttranslocated state rectifies the forward motion in a mechanism that is consistent with Pol II operating as a Brownian ratchet (1-3). After the binding of an NTP, Pol II rapidly hydrolyzes the NTP, extends the nascent RNA transcript by 1 nt, and releases pyrophosphate (PPi). During transcription elongation, Pol II is also susceptible to entering a paused state, which is a major regulatory element for transcriptional repression (4). In a paused state, Pol II is known to backtrack wherein the 3′ end of the nascent RNA transcript is extruded from its active site. Backtracked Pol II molecules remain catalytically inactive and may become transcriptionally competent only when the enzyme restores the registry between the Pol II's active site and the 3′ end of the transcript either by diffusion forward along the DNA template or by cleaving of the misaligned transcript at the backtracked position of the active site (1, 5). Many transcription factors, including TFIIS and TFIIF, are known to regulate transcription elongation by directly interacting with the polymerase (6). TFIIS rescues backtracked Pol II molecules by stimulating the intrinsic endonucleolytic activity of Pol II (7). An internal scission of the RNA backbone removes 2-nt or longer fragments of the nascent RNA and returns the enzyme to a posttranslocated state, from which it resumes transcription elongation (8). Misincorporated nucleotides favor backtracking of the enzyme; thus, TFIIS-induced cleavage promotes transcription fidelity both in vitro and in vivo (9-11). TFIIF, by contrast, has an established role in transcription initiation, where it associates with Pol II and five other general transcription factors to form the preinitiation complex. TFIIF is necessary for the recruitment of Pol II to the preinitiation complex, and it either recruits or retains TFIIB during transcription initiation (12-15). A recent cryo-EM reconstruction of the preinitiation complex of human Pol II suggests that TFIIF can stabilize the downstream DNA along the cleft of the enzyme (16), in addition to stabilizing the RNA-DNA hybrid within the polymerase (14). TFIIF is also involved in the elongation phase of transcription in vivo in both yeast (17) and mammals (15). TFIIF is known to stimulate the overall elongation rate in mammalian systems (18) (19) (20) ; however, the detailed mechanism by which TFIIF affects the elongation phase is still unknown. Moreover, although TFIIF is known to bind elongating Pol II in yeast (17) , its effects on the elongation process have not been demonstrated. Transcription elongation in eukaryotic cells is also regulated by the presence of histones that organize the DNA in the form of Significance Regulation of gene expression controls fundamental cellular processes, such as growth and differentiation. Gene expression begins at transcription, in which the eukaryotic RNA polymerase (Pol II) synthesizes the precursors of mRNA during the elongation phase. The speed and fidelity of the process are regulated by many transcription elongation factors, including transcription factors IIS (TFIIS) and IIF (TFIIF), which directly interact with the enzyme. Using a single-molecule optical-tweezers assay, we have quantified the effects of these factors on the dynamics of transcription elongation by Pol II on both bare and nucleosomal DNA. We showed that TFIIF mainly prevents Pol II from pause entering, whereas TFIIS assists with pause recovery, and quantitatively described these effects on the kinetics of transcription elongation by Pol II.
doi:10.1073/pnas.1401611111 pmid:24550488 pmcid:PMC3948276 fatcat:ljr3ypikkrazha3d2yvzb2yzay