Defining mechanisms that regulate RNA polymerase II transcription in vivo

Abstract

In the eukaryotic genome, the thousands of genes that encode messenger RNA are transcribed by a molecular machine called RNA polymerase II. Analysing the distribution and status of RNA polymerase II across a genome has provided crucial insights into the long-standing mysteries of transcription and its regulation. These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step. When coupled with genome-wide mapping of transcription factors, these approaches identify key regulatory steps and factors and, importantly, provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.

Figure 1. Transcription regulatory interactions

General transcription factors (GTFs) bind to specific sequence elements in the promoter. These elements (the B recognition element (BRE), the TATA box (TATA), the initiator (Inr), the motif ten element (MTE) and the downstream promoter element (DPE)) and their approximate locations relative to the transcription start site (TSS, black arrow) are shown. Transcriptional regulators (orange oval and yellow diamond), which are either activators or repressors, bind to specific DNA sequences located near the core promoter of the gene or various distant regions, called enhancers. The regulators can interact (green arrows) with GTFs, such as TFIID (blue rectangle) and TATA-binding protein (TBP, blue horseshoe), and the Pol II complex (red ‘rocket’) to enhance or repress transcription. They also interact (green arrows) with co-regulators (green hexagon) that can interact (blue arrows) with the general transcription machinery or chromatin-modifying factors, such as histone modifiers or nucleosome remodellers. The co-regulators can also bind to nucleosomes (green) with various histone modifications, stabilizing the co-regulator binding to the gene. Activators can recruit, stabilize or stimulate these factors, and repressors can disrupt or inhibit these factors.

Figure 2. The transcription cycle is a multistep process

Step 1: chromatin opening. The repressed gene and regulatory region are entirely packaged as nucleosomes (green). An activator (orange oval) binds and recruits nucleosome remodellers to clear the promoter. Step 2: PIC formation. A second activator (yellow diamond) binds, promotes the binding of GTFs (blue rectangle) and recruits coactivators (green hexagon), facilitating Pol II (red rocket) entry to the PIC. Step 3: initiation. DNA is unwound (oval inside Pol II) at the TSS, and an open complex is formed. Step 4: promoter escape/clearance. Pol II breaks contacts with promoter-bound factors, transcribes 20–50 bases downstream of the TSS, produces an RNA (purple line) and pauses, partially mediated by SPT4–SPT5 in Drosophila (pink pentagon) and negative elongation factor (NELF) complex (purple circle). The Ser residues at position 5 (Ser 5) of the Pol II carboxy-terminal domain (CTD) repeats are phosphorylated (red P) during this step. Step 5: escape from pausing. P-TEFb (blue triangle) is recruited directly or indirectly by the activator and phosphorylates Ser 2 of the Pol II CTD repeats, SPT5 and the NELF subunits (blue Ps). NELF dissociates from the rest of the complex. Pol II escapes from the pause, either terminating or entering productive elongation. Step 6: productive elongation. Nucleosomes are disassembled and reassembled as the Pol II elongation complex transcribes through the gene. Step 7: termination. After the Pol II complex transcribes the gene, it is removed from the DNA, and the RNA is released. Step 8: recycling. The freed Pol II can reinitiate.