What does rna polymerase 2 do

Sequence-specific, DNA-binding TFs drive all biological processes Lee and Young and they function in part by binding enhancer or promoter sequences and subsequently recruiting the PIC to specific genomic loci. Among the few structurally characterized TF-Mediator interfaces Yang et al. Structured, hydrophobic pockets represent druggable targets that could be exploited for therapeutic purposes.

Along these lines, Arthanari and coworkers Nishikawa et al. Furthermore, several laboratories have designed small molecules that mimic transcriptional activators, presumably through binding Mediator—TF interfaces Rowe et al.

PIC structural models that include the Mediator complex. Colors for each PIC factor are identical to Figure 1. Structural remodeling is likely upon binding TFIIH, as clashes are evident in this artificially docked model. The differences between yeast see B and human Mediator reflect the much larger size of the human Mediator complex Table 1. These differences could result from true differences in PIC structure yeast vs.

Here, a core Mediator complex is shown in green, whereas all other PIC factors are shown in the same colors as A. The different orientation of downstream DNA vs. A reflects potential structural differences between yeast and human PICs.

Note, however, that A is a hypothetical model that merges two different structures, whereas B represents cryoEM data from a single structure Schilbach et al.

Adapted from PDB 5oqm Schilbach et al. Such structural transitions may be highly dependent on the MED14 subunit Cevher et al. A recent cryoEM analysis of Mediator isolated from murine B cells expands upon these concepts and has provided the highest resolution 5.

As expected, the mouse Mediator complex showed some structural distinctions with yeast Mediator based on its larger size. For instance, the various mobile domains appeared to be more interconnected in the mouse Mediator complex, suggesting that potential conformational changes may require more extensive remodeling of protein—protein interfaces compared with yeast.

Subunits comprising the tail segment Med15, Med16, Med23—25, and Med27—30 were also shown to be more structurally integrated vs. Whereas structures of S. A review focused on Mediator structure and function, based largely on cryoEM structural data, was recently published Harper and Taatjes ; below, we highlight new cryoEM results with a yeast complex associated with the PIC Fig.

The Cramer laboratory Schilbach et al. This structure Schilbach et al. Comparison of the free cMED structure from S. This observation is consistent with lower-resolution cryoEM data with human Mediator Fig. Med6 and Med7 occupy the shoulder and knob regions of Mediator, respectively Nozawa et al.

These results are consistent with established roles for Mediator in stabilizing PICs in yeast Eyboulet et al. Because intermediate- to high-resolution structural data are lacking for mammalian Mediator-PIC assemblies, it remains to be determined whether specific molecular interfaces will be conserved.

At this point, the Pol II clamp closes slightly He et al. Collectively, these interactions between the PIC and promoter DNA require precise positioning at the time of promoter melting. The large size of the PIC likely provides the structural stability necessary to properly orient each of these domains in 3D space, along and within the Pol II cleft.

After transcription initiation, additional structural transitions must occur once the nascent RNA reaches a length of 12—13 nt. Structural data from the Cramer laboratory Vos et al. These so-called elongation factors do not represent an exhaustive list of proteins and protein complexes that control Pol II transcription, but each relates back to PIC factors in various ways.

Moreover, the factors described below are implicated in early stages of transcription i. Nucleophilic cleavage of the RNA is likely mediated by an activated water molecule, with the acidic residues involved in positioning metal ions to promote cleavage of the phosphodiester bond. Furthermore, RNA backtracks into the funnel, and elongation is blocked. Such backtracked Pol II enzymes represent stably paused intermediates and may require polyubiquitination and degradation to remove from the DNA template Sigurdsson et al.

Based on in vitro Missra and Gilmour ; Li et al. This is supported by cell-based studies that suggest reduced capacity to transcribe long genes if DSIF function is disrupted Shetty et al.

This site is also adjacent to the upstream DNA exit and therefore may promote reannealing of the DNA template behind the transcribing polymerase Bernecky et al. This could be important to prevent formation of R-loops, which could otherwise disrupt transcription and contribute to genome instability Sollier et al.

The CTR9 subunit contains several interesting structural features. These may play important roles in promoting transcription through chromatin, consistent with the function for the PAF complex Pavri et al.

An N-terminal region of SPT6 residues 1— was disordered in the complex but is positioned such that it could potentially interact with nucleosomes in front of or behind the transcribing polymerase, consistent with its biological roles in RNA processing and as a histone chaperone Bortvin and Winston ; Kaplan et al. This coincided with a movement of upstream DNA i.

Collectively, these structural transitions may increase the rate of Pol II elongation by facilitating RNA exit and promoting DNA reannealing behind the transcribing polymerase. Crystal structures of the complex have been determined Baumli et al.

These studies revealed dozens of high-confidence targets, with many representing transcription cofactors or RNA processing factors. Using biochemical assays and cryoEM, the Cramer laboratory Vos et al.

RNA processing e. The Mediator kinase module is a large complex kDa; Table 1 that contains four subunits Fant and Taatjes In the yeast S. Low-resolution cryoEM structures of the entire yeast and human kinase modules have been determined Knuesel et al.

No structural data exist for these paralogs. Biochemical studies suggested that the N terminus of MED12, which is a hot spot for oncogenic mutations Makinen et al. Similar activation loops also known as T-loops exist in other CDKs, but the CDK8 sequence contains a D instead of a typical T at residue , suggesting a phosphorylation-independent activation mechanism.

The structural discrepancies between these models for MEDdependent activation of CDK8 could reflect two distinct mechanisms of activation. Future experiments will benefit from analysis of complete CDK8 module assemblies or evaluation with knock-in cell lines that ensure expression of mutant subunits at physiologically relevant levels.

These results suggest that the Mediator kinase module may function at postinitiation stages of Pol II transcription, and this is supported by cellular and biochemical data Donner et al. Gene expression changes are markedly different upon CDK8 kinase inhibition compared with subunit knockdown Poss et al. Structural data from cryoEM and complementary methods, combined with functional studies, has advanced our understanding of Pol II transcription. Twenty years ago, the set of PIC factors had been identified but the basic structural architecture of the PIC was not known.

At present, we have a greatly improved understanding of PIC structure, function, and dynamics. Many regulatory interfaces have been identified at high resolution, which allows rational design of molecular probes for mechanistic studies or as lead compounds for molecular therapeutics. However, key details remain to be uncovered and we focus on a number of outstanding questions below.

The size Table 1 and flexibility of these factors contributes to the difficulties in understanding their structural and functional roles.

For instance, the Nogales laboratory Greber et al. This positions the kinase module more favorably for Pol II CTD phosphorylation, but it remains unclear how this repositioning is triggered or whether the TFIIH kinase module remains anchored at this site during transcription.

It is also unclear how transcription reinitiation is regulated by the PIC. In vitro, reinitiation has been shown to occur more rapidly compared with de novo initiation Hawley and Roeder ; Jiang and Gralla It was proposed by Nogales et al. Patel et al. Conceptually, this is similar to known roles for paused Pol II in the maintenance of nucleosome-free regions at active gene promoters Gilchrist et al.

In cells, a phenomenon called transcriptional bursting has been described Fukaya et al. Transcriptional bursting appears to involve rapid reinitiation by multiple Pol II enzymes, but the molecular mechanisms remain unknown Lenstra et al. Throughout the stages of PIC assembly, initiation, promoter escape, pausing, and elongation, numerous factors compete for the same binding surfaces on Pol II.

Consequently, regulation of these interactions is paramount. Although the mutually exclusive binding of initiation versus elongation factors provides a biological rationale for their exchange during different transcriptional stages, Pol II transcription will not be efficient if initiation, pausing, or elongation factors continually compete for Pol II binding. Precisely how these interactions are controlled remains incompletely understood.

Consistent with this, CTD phosphorylation promotes formation of condensates that exclude Mediator Guo et al. Another means of regulation is through posttranslational modifications, such as phosphorylation by transcription-associated kinases. Although this is complicated by the array of kinases and phosphatases that can converge at sites of active transcription, it is well-established that phosphorylation can increase or decrease protein-protein or protein-nucleic acid binding affinities Pufall et al.

Collectively, dynamic and reversible modification of proteins through posttranslational modifications or segregation of initiation versus elongation factors into biophysically distinct molecular condensates could help ensure that initiation, pausing, and elongation factors interact with Pol II at the appropriate stages of transcription. Note that these potential regulatory mechanisms are not mutually exclusive and may function cooperatively throughout transcription initiation, elongation, and termination.

Much remains to be discovered about the structural transitions that the PIC undergoes during transcription initiation. Potentially, these TF-induced structural changes could contribute to transcriptional bursting, given that transient TF—DNA binding has been shown to correlate with bursting Mir et al.

Details about the molecular mechanisms await higher-resolution information for the Mediator—Pol II structural transitions that result in transcription activation. Evidence for TFIID conformational changes during transcription initiation have been obtained through biochemical studies Yakovchuk et al. Similarly, several distinct Mediator—Pol II structural intermediates are likely to have functional relevance during Pol II initiation and promoter escape, based on the extensive interaction between these complexes and the demonstrated conformational flexibility Bernecky et al.

CryoEM is suited to address these challenges and multiple functionally distinct intermediates could be characterized through a combination of biochemical and computational approaches. Finally, a detailed mechanistic understanding of Pol II transcription will require characterization of PIC dynamics in real time.

In vitro single molecule studies Tomko and Galburt can augment structural data to better define how Pol II and associated regulatory factors work together to transcribe from a DNA template. Furthermore, advances in live cell imaging will complement the continually improving structural and mechanistic models of Pol II transcription.

Despite recent progress Liu and Tjian , many basic questions remain unanswered, such as 1 how genomes are organized in the three-dimensional space of the nucleus Furlong and Levine , 2 how transcriptional bursting occurs Donovan et al.

Also, what set of cofactors are essential for these processes, and which are redundant, context-specific, or cell type-specific? Addressing these questions will be important but challenging, especially in mammals, which have larger genomes, more elaborate enhancer—promoter regulatory networks Levine et al.

Fortunately, given the technological and methodological advances over the past few decades, we have reached a point at which most experimental questions can be rigorously addressed. View all Schier and Dylan J. Previous Section Next Section. Figure 1. Figure 2. View this table: In this window In a new window. Table 1. Figure 3. The trigger loop and bridge helix The trigger loop moves between an open and closed state with each NTP addition cycle Kaplan et al. The cleft, clamp, and stalk The cleft and stalk are the most prominent Pol II structural features.

The wall and protrusion The wall resides in the cleft, near the active site, and represents the site at which the RNA:DNA hybrid separates.

Figure 4. Figure 5. Figure 6. Mediator kinase module The Mediator kinase module is a large complex kDa; Table 1 that contains four subunits Fant and Taatjes Previous Section. Mol Cell 17 : — Transcription without XPB establishes a unified helicase-independent mechanism of promoter opening in eukaryotic gene expression.

Mol Cell 65 : — CrossRef Google Scholar. Nat Struct Mol Biol 20 : — CrossRef Medline Google Scholar. Andel F 3rd. Science : — Nat Struct Mol Biol 25 : — Genes Dev 33 : — Genes Dev 8 : — A kinase-independent role for cyclin-dependent kinase 19 in p53 response. Mol Cell Biol 37 : e Nucleic Acids Res 47 : — EMBO J 27 : — Decreased enhancer—promoter proximity accompanying enhancer activation.

Mol Cell 76 : — Bernecky C , Taatjes DJ. J Mol Biol : — PLoS Biol 9 : e Nature : — Nat Struct Mol Biol 24 : — Proc Natl Acad Sci : — RNA polymerase II clustering through carboxy-terminal domain phase separation. J Biol Chem : — Bortvin A , Winston F. Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Brueckner F , Cramer P.

Nat Struct Mol Biol 15 : — Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. Nat Struct Mol Biol 21 : — EMBO J 29 : — Cheung AC , Cramer P. Cell : — Proc Natl Acad Sci 88 : — Core L , Adelman K. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers.

Nat Genet 46 : — Nucleic Acids Res. Shi, Y. Egloff, S. Buratowski, S. Cell 36 , — The CTD code. Gromak, N. Shows that a pause sequence promotes poly A -dependent termination in vivo , and the efficiency of termination is influenced by the strength of the poly A site and its proximity to the pause site. Glover-Cutter, K. Park, N. The two steps of poly A -dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain.

Nag, A. The poly A -dependent transcriptional pause is mediated by CPSF acting on the body of the polymerase. Kazerouninia, A. Poly A signal-dependent degradation of unprocessed nascent transcripts accompanies poly A signal-dependent transcriptional pausing in vitro.

RNA 16 , — References 33—35 from the Martinson laboratory show that poly A -dependent termination of Pol II can be separated into two steps, pausing and release, which depend on interactions of the cleavage and polyadenylation machinery with the body of Pol II and the Pol II CTD. Alexander, R. Splicing-dependent RNA polymerase pausing in yeast. Carrillo Oesterreich, F.

Global analysis of nascent RNA reveals transcriptional pausing in terminal exons. Kim, M. Nature , — Luo, W.

The authors propose a model for the termination mechanism that incorporates both allosteric and torpedo components. Lunde, B. Teixeira, A. Ghazal, G. Cell 36 , 99— Fail-safe transcriptional termination for protein-coding genes in S.

Cell 36 , 88—98 Nabavi, S. FEBS Lett. Connelly, S. Houseley, J. The many pathways of RNA degradation. Biochemistry 38 , — Steinmetz, E. Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1. Cell 24 , — Ursic, D. Finkel, J. Sen1p performs two genetically separable functions in transcription and processing of U5 small nuclear RNA in Saccharomyces cerevisiae.

Genetics , — Mischo, H. Yeast Sen1 helicase protects the genome from transcription-associated instability.

Cell 41 , 21—32 Arigo, J. Cell 23 , — Thiebaut, M. Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the Nrd1-Nab3 pathway in genome surveillance. Banerjee, S. Rho-dependent transcription termination: more questions than answers. Kawauchi, J. Banerjee, A.

Suraweera, A. Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation. Matera, A. Cell Biol. Science , — Ezzeddine, N. Baillat, D. Dominski, Z. Ballarino, M. Carninci, P. Molecular biology: the long and short of RNAs. Dengl, S. Saeki, H. Stability, flexibility, and dynamic interactions of colliding RNA polymerase II elongation complexes. Cell 35 , — Xiang, S. Chang, J. Nature Cell Biol. Epshtein, V. An allosteric mechanism of Rho-dependent transcription termination.

Demonstrates that the Rho termination factor associates directly with E. Lang, W. USA 95 , — Schmidt, M. Sullivan, S. Requirement for E. Cell 68 , — Mason, S. Torres, M. Ribosomal protein S4 is a transcription factor with properties remarkably similar to NusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination. Shankar, S. A transcription antiterminator constructs a NusA-dependent shield to the emerging transcript.

Cell 27 , — Ha, K. Toulokhonov, I. Deighan, P. USA , — Mooney, R. Two structurally independent domains of E. Reveals how interactions with two separate protein domains of NusG contribute to its RNA polymerase termination and antitermination activities.

Nickels, B. Genetic assays to define and characterize protein—protein interactions involved in gene regulation. Methods 47 , 53—62 USA 88 , — This publication describes how unphosphorylated CTD is associated with the transcription pre-initiation complex. Robinson, P. Cell , — Hahn, S. Transcriptional regulation in Saccharomyces cerevisiae : transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators.

Payne, J. The transition of RNA polymerase II from initiation to elongation is associated with phosphorylation of the carboxyl-terminal domain of subunit IIa. Wong, K. Cell 54 , — Kin28 regulates the transient association of Mediator with core promoters.

Hyperphosphorylation of the C-terminal repeat domain of RNA polymerase II facilitates dissociation of its complex with mediator. Cadena, D. Descostes, N. Mayer, A. Science , — Schroeder, S.

Licatalosi, D. Cell 9 , — Hintermair, C. EMBO J. McCracken, S. Kim, M. Phosphorylation of the yeast Rpb1 C-terminal domain at serines 2, 5, and 7.

Nature , — This study identifies how general transitions in the transcription process are coupled to changes in CTD phosphorylation. Kim, H. Tietjen, J. Chemical-genomic dissection of the CTD code.

Bataille, A. Cell 45 , — Adelman, K. Wu, C. Jonkers, I. Cell Biol. Ni, Z. Peterlin, B. Controlling the elongation phase of transcription with P-TEFb. Cell 23 , — Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. Li, J. Venkatesh, S. Histone exchange, chromatin structure and the regulation of transcription. Sun, M. Fuchs, S. Protein modifications in transcription elongation.

Acta , 26—36 Devaiah, B. Chen, F. Yu, M. Cell 63 , — Liu, J. Qiu, H. Yoh, S. Bartkowiak, B. Liang, K. Blazek, D. Acta , — Bentley, D. Coupling mRNA processing with transcription in time and space. Baranello, L. RNA polymerase II regulates topoisomerase 1 activity to favor efficient transcription. Cell 51 , — Chen, C. Ho, C. Cell 3 , — Cooke, C. Flaherty, S. USA 94 , — RNA 2 , — Topisirovic, I. Cap and cap-binding proteins in the control of gene expression.

Carrillo Oesterreich, F. CAS Google Scholar. Warner, J. The economics of ribosome biosynthesis in yeast. Tardiff, D. A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly posttranscriptional. Cell 24 , — Moore, M. Differential recruitment of the splicing machinery during transcription predicts genome-wide patterns of mRNA splicing. Global analysis of nascent RNA reveals transcriptional pausing in terminal exons.

Cell 40 , — David, C. Gu, B. Nucleic Acids Res. Morris, D. Milligan, L. Nojima, T. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Ng, H. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Cell 11 , — Krogan, N. Lee, J. Wdr82 Is a C-terminal domain-binding protein that recruits the Setd1A histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes.

Terzi, N. Li, B. The Set2 histone methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II. This work reveals that co-transcriptional histone methylation is coupled to transcription by the CTD. Xiao, T. Schaft, D. The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation. Kizer, K. Vojnic, E.