Pol II Accumulation

Excerpts from "Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing"

"Significant accumulation of Pol II over the 3’ cleavage/polyA region of genes is proposed to facilitate 3’ processing and transcription termination(9,10). Finally, the interplay of transcription rate and splicing efficiency(11) might be reflected in the selective accumulation of Pol II at splice junctions."

"Protein factors such as Negative Elongation Factor (NELF) and DRB Sensitivity Inducing Factor (DSIF)(3,13), DNA elements(14,15), DNA sequence composition(16), nascent RNA processing(16), and nucleosomes(17) can influence pausing."

"ChIP-based methods that collect Pol II or associated RNAs do not distinguish paused Pol II from other Pol II-RNA complexes(16,18,19). The genome-wide nuclear run-on approach (GRO-seq method)(6–8) circumvents these issues by enriching nascent transcripts only associated with actively engaged polymerase with high sensitivity, but it has a resolution of only 30–50 bases(18)."

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Excerpts from "Pol II waiting in the starting gates: regulating the transition from transcription initiation into productive elongation"

"Recognition of promoters begins with the assembly of a large protein complex containing Pol II and multiple General Transcription Factors (GTFs) on the promoter. The minimal set of factors required for the formation of this pre-initiation complex (PIC) includes Pol II, the GTFs TFIIB, TFIID (which includes the TATA-binding protein, TBP), TFIIE, TFIIF and TFIIH. Extensive interactions between the polymerase and GTFs increase the affinity of Pol II for the promoter region. In addition to the GTFs, recruitment of Pol II to promoters is greatly influenced by the Mediator complex, DNA-binding transcription activators, and a vast repertoire of nucleosome remodeling and modifying complexes (reviewed in [16, 17])."

"While the exact mechanisms of TSS selection by Pol II are not completely clear, its positioning on the promoter may largely depend on the sequence specificity of GTF interactions with promoter DNA. Indeed, while transcription initiation from promoters that contain distinct sequence elements such as the TATA box, Initiator, or Downstream Promoter Element (DPE) is often very focused and likely to arise from a single nucleotide position, initiation from promoters that lack these motifs is much more dispersed (reviewed in [18])."

Distinctions between poised/paused/backtracked/arrested/stalled polII was also made in this review.

Experimental techniques in detecting paused polymerase:
ChIP: "Low spatial resolution, complicating the distinction between engaged Pol II and polymerase in pre-initiation complexes. ChIP may be difficult to perform in samples that are not in a homogeneous suspension, such as tissues."

Permanganate reactivity: "Detects locally melted regions of DNA, including those arising from paused polymerase, by selectively modifying unpaired Thymines within a stable, open transcription bubble. Advantages: Can be performed directly on whole cells or tissues. Achieves essentially nucleotide-level resolution for mapping paused polymerase. Does not require antibodies. Disadvantages: Low throughput (requires Ligation-Mediated (LM) PCR on individual genes - no genome-wide application as of yet). Application is limited to genes where good primers for primer extension and LM PCR can be designed; because of that, permanganate probing in mammalian systems has been markedly less successful than in Drosophila. Since actively elongating polymerase generates very transient melting of DNA at a given location, permanganate is inefficient at detecting productive elongation complexes."

Nuclear run-on: "Detects elongation-competent RNA polymerases through their ability to incorporate a label into nascent RNA. Advantages: Specifically reveals transcriptionally engaged polymerase, with extremely high sensitivity and low background. Adaptable for high-throughput genome-wide applications. Can be used in various organisms. Disadvantages. Requires preparation of nuclei to detect paused polymerase. Resolution for mapping of paused polymerase is reduced by the necessity to allow polymerase to run-on and incorporate labeled nucleotides into RNA."

RNA analysis: "Directly detects short RNA species derived from paused Pol II. Advantages. Sequencing of RNAs from the 3′-end reveals the positions of promoter-proximally paused polymerase at nucleotide-level resolution. Designed for high-throughput genome-wide applications. Does not require antibodies, cell treatment or labeling. Can be used in various organisms. Disadvantages. Requires enzymes available from only one commercial source. Cannot distinguish between RNA species that remain associated with paused Pol II and those that have been released."