Efficient and Rapid Nucleosome Traversal by RNA Polymerase II Depends on a Combination of Transcript Elongation Factors

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FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/ H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.elongation | dynamics | DNA uncoiling | mechanism F ACT (facilitates chromatin transcription) is the transcription and replication factor (1, 2) involved in cell differentiation (3), and is also an important target for anticancer drugs (4). Human FACT (hFACT) is a heterodimer protein complex composed of two subunits [suppressor of Ty 16 homolog (Spt16) and structure specific recognition protein 1 (SSRP1)] that has histone chaperone activity (5, 6). FACT stimulates transcript elongation through nucleosomes in vitro (2, 6). In vivo, FACT colocalizes with RNA polymerase II (Pol II) and displays similar kinetics of recruitment and chromosome tracking (7,8). FACT is also essential for maintenance of chromatin structure during transcript elongation by Pol II (8-11).Different models have been proposed to describe the mechanism of FACT's action. FACT can induce global accessibility of nucleosomal DNA without histone H2A/H2B displacement (12, 13) and thus can facilitate action of processive enzymes on DNA. On the other hand, it has been suggested that FACT destabilizes nucleosomes by facilitating dissociation of histone H2A/H2B dimer from nucleosomes, thereby facilitating transcription through chromatin (6). The mechanism of FACT action during transcription remains to be experimentally evaluated.In this study, we systematically examined the effect of FACT on transcription through a nucleosome by Pol II in vitro. Our results show that FACT alleviates nucleosomal pausing, and that the presence of H2A/H2B dimers is required for FACT action. Kinetic studies suggest that the alternating FACT-dimer interactions result in an increased rate of conversion from the nonproductive to productive Pol II-nucleosome complexes and thus facilitate transcription and nucleosome survival. ResultsH2A/H2B Dimers Mediate FACT-Dependent Transcription Through a Nucleosome. In our experiments, we used yeast Pol II and DNA fragments bearing single nucleosomes assembled on DNA sequences having high affinity for the...

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 98%

Histone chaperone FACT action during transcription through chromatin by RNA polymerase II

Hsieh¹,

Kulaeva

Patel³

et al. 2013

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224

“…These factors are believed to work in concert to enhance the efficiency of productive elongation (14)(15)(16)(17). TFIIS is known to enhance the intrinsic endonucleolytic cleavage activity of RNAPII, leaving a fresh 3′ end of the RNA aligned with the active site, which allows transcription to resume after any backtracking and facilitates multiple attempts to overcome transcriptional barriers (7,10,18,19).…”

mentioning

confidence: 99%

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms

Schweikhard¹,

Meng

Murakami³

et al. 2014

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule opticaltrapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.Pol II | optical tweezers | optical trap T he expression of most genes is carefully regulated at the level of transcription. As a consequence, RNA polymerase II (RNAPII)-the enzyme responsible for mRNA synthesis in eukaryotic organisms-is at the nexus of an exquisite network of regulatory pathways, many of which are controlled by transcription factors. The control pathways associated with RNAPII recruitment to, and initiation at, promoter sites have been studied extensively (1), but it has become increasingly clear that significant regulatory activity also occurs during postinitiation steps and, in particular, at the level of transcript elongation (2).Productive transcript elongation (3-5) is characterized by periods of unidirectional motion by RNAPII along the DNA template, adding one nucleotide at a time to the growing RNA transcript. Elongation-both in vitro in highly purified systems (6, 7) and in vivo (8, 9)-is frequently interrupted by transcriptional pauses, at least some fraction of which are associated with enzyme backtracking, a process by which RNAPII reverses its normal direction of motion and moves upstream on the template (6, 7). Entry into backtracked states appears to confer a high degree of for...

“…Release of TFIIB from the elongation complex (EC) is reported to begin 7 to 16 bases downstream of transcription start (13). TFIIF should remain associated with the EC to facilitate rapid and effective transcript elongation (14,15), but this factor may be transiently lost after initiation (16).…”

mentioning

confidence: 99%

Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes

Čabart

Újvári

Pal

et al. 2011

Self Cite

Transcription factors TFIIB and TFIIF are both required for RNA polymerase II preinitiation complex (PIC) assembly, but their roles at and downstream of initiation are not clear. We now show that TFIIF phosphorylated by casein kinase 2 remains competent to support PIC assembly but is not stably retained in the PIC. PICs completely lacking TFIIF are not defective in initiation or subsequent promoter clearance, demonstrating that TFIIF is not required for initiation or clearance. Lack of TFIIF in the PIC reduces transcription levels at some promoters, coincident with reduced retention of TFIIB. TFIIB is normally associated with the early elongation complex and is only destabilized at +12 to +13. However, if TFIIF is not retained in the PIC, TFIIB can be lost immediately after initiation. TFIIF therefore has an important role in stabilizing TFIIB within the PIC and after transcription initiates.general transcription factors | transcript initiation P romoter-directed transcript initiation by RNA polymerase II (pol II) on double-stranded templates requires at minimum the general transcription factors TBP (TATA box-binding protein), TFIIB, TFIIF, TFIIE, and TFIIH. Genetic and biochemical evidence indicates that TFIIB can direct transcription start site selection (1-4) with the assistance of TFIIF (5-8). When TFIIB is in complex with pol II, a TFIIB domain referred to as the finger (9) or reader (10) enters pol II and approaches the catalytic center. Structural investigations of the yeast preinitiation complex (PIC) revealed a segment of TFIIF localized near the TFIIB domain that presumably directs transcription start site selection (7,11,12). It was suggested that this interaction with TFIIF is important in stabilizing TFIIB within the PIC (12). Release of TFIIB from the elongation complex (EC) is reported to begin 7 to 16 bases downstream of transcription start (13). TFIIF should remain associated with the EC to facilitate rapid and effective transcript elongation (14, 15), but this factor may be transiently lost after initiation (16).The functional roles of TFIIB and TFIIF within the PIC are not fully understood. Transcription from a preformed transcription bubble required only TFIIB and TBP, although TFIIF was strongly stimulatory in that system (17). RNA synthesis from bubble templates in the presence of the complete set of general factors was also very strongly dependent on TFIIF (18). These results raise an important question: Do TFIIB and TFIIF participate in the formation of initial bonds, or are they simply structural components that direct pol II to the correct start site? An important related issue concerns the continued residence of these factors within the EC: Is the proposed loss of TFIIF after initiation linked to the expected release of TFIIB?In the present paper we take advantage of our recent observation that phosphorylation of TFIIF with casein kinase 2 (CK2) abolishes the ability of TFIIF to associate with pol II in ECs and stimulate elongation effectively (19), while leaving TFIIF's support of tra...