Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pausefree velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtain direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for regulation of eukaryotic transcription.During transcription elongation in eukaryotes, RNA polymerase II must overcome the transcriptional barriers imposed by nucleosomes in chromatin. In vitro, a single nucleosome is sufficient to halt or greatly slow transcription by Pol II (1-5), and factors that restrict transcriptional backtracking relieve nucleosome-induced pauses and arrests, suggesting that the influence of the nucleosome is mediated through polymerase backtracking (4). Pol II also affects nucleosomal dynamics: depletion and turnover of histones are seen in actively transcribed genes in vivo (6,7), and histones are often transferred behind transcribing polymerases in vitro (2). However, the mechanisms underlying the mutual influence between nucleosome and polymerase are not well understood.Here a dual-trap optical tweezers assay revealed real-time trajectories of individual Pol II complexes as they transcribed through single nucleosomes. A tether was created between two trapped beads-one attached to a stalled polymerase, the other to the upstream DNA ( Figure 1a and Supporting Material). Addition of ribonucleotide triphosphates induced the polymerase to move towards the nucleosomal positioning sequence (NPS), causing the force between the * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the

RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked. Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3 end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme. The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage. Secondary structure elements or the 5 end of the transcript can prevent the isomerization by blocking the RNA threading. The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.Arrested ternary complexes of prokaryotic and eukaryotic RNA polymerases (RNAPs) retain the RNA but lose catalytic activity (1-4). DNA sites that provoke arrest do not show strong sequence similarity except that they are enriched in homopolymeric oligo(T) tracts that are 7-10 nucleotides long (4-6). However, transcription through certain regions in DNA is particularly predisposed to arrest. At such sites, the front end of the RNAP footprint appears to remain fixed on the template while RNA chain growth continues (5, 7-10). These unusual regions were called sites of discontinuous elongation, because normally RNAP footprints translocate monotonously along the template as each nucleotide is added to the RNA.Being a potential block for RNAP passage through a transcription unit, arrest may affect gene expression by decreasing formation of full-sized transcripts, as documented in vitro for Escherichia coli RNAP transcribing the rrnB gene (3) and for eukaryotic RNA polymerase II (Pol II) transcribing histone H3.3, and several other genes (3, 4, 11). In addition, arrested complex formation represents a side pathway in normal termination, since a fraction of the ternary complexes at prokaryotic and intrinsic Pol II termination sites fall into an arrested state instead of dissociating from the template (4, 5).Elongation factors TFIIS in eukaryotes and GreB in prokaryotes relieve arrest by inducing internal cleavage of the transcript (3,6,(12)(13)(14)(15). The 3Ј-proximal product of the cleavage may reach 10-20 nucleotides in length, whereas in active complexes the analogous products are only 2-3 nucleotides long (3,6,16,17). The cleaved RNA fragments dissociate from the complex, which allows RNAP to resume elongation in the correct DNA-RNA register from the newly formed 3Ј end. No movement of RNAP was detected upon transcript cleavage in arrested complexes (18), whereas in the majority of active complexes transcript cleavage caused upstream shifting of the enzyme (9, 19).Many studies of arrest were performed with artificially halted individual elongation complexes (ECs) obtained by ''walking'' the RNA...

RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo. We examined transcription by Pol II through nucleosome cores in vitro. At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.

In the cell, RNA polymerase II (pol II) efficiently transcribes DNA packaged into nucleosomes, but in vitro encounters with the nucleosomes induce catalytic inactivation (arrest) of the pol II core enzyme. To determine potential mechanisms making nucleosomes transparent to transcription in vivo, we analyzed the nature of the nucleosome-induced arrest. We found that the arrests have been detected mostly at positions of strong intrinsic pause sites of DNA. The transient pausing makes pol II vulnerable to arrest, which involves backtracking of the elongation complex for a considerable distance on DNA. The histone-DNA contacts reestablished in front of pol II stabilize backtracked conformation of the polymerase. In agreement with this mechanism, blocking of backtracking prevents nucleosome-induced arrest. Transcript cleavage factor TFIIS reactivates the backtracked complexes and promotes pol II transcription through the nucleosome. Our findings establish the crucial role of elongation factors that suppress pol II pausing and backtracking for transcription in the context of chromatin.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.