RNA Polymerase II

Introduction

RNAP II transcription process.

RNA polymerase II (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA. There are three RNA polymerase enzymes found in eukaryotic nuclei but RNAP II is the most studied. RNAP II is a 550 kDa multi-protein complex that includes 12 subunits. Several transcription factors are used to bind promoters upstream of the start site and are necessary for joining RNAP II and DNA. Bound RNAP II transcribes DNA into a strand of messenger RNA. Messenger RNA (mRNA) is a single stranded RNA molecule that is complementary to the template strand of DNA. The mRNA stand transports genetic information from DNA to the ribosome, where it is used to specify the amino acid sequence for the production of proteins.

Structural Components

This section will briefly discuss the chief structural components involved in the mechanism.

The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.

To begin, the swings to trap the DNA in the cleft. Further along, the sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II.

Other components of RNA pol II include the following:

The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. is located within the active site and functions as the catalyst.

Mechanism of Action

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α-Amanitin

α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover.

The chemical structure of α-amanitin.

Modifications

General Transcription Factors

In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.

Process of PIC formation:

1. contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.

2. and and interact with TBP and are recruted to the promoter.

3. binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly.

4. and are sequentually recruited which completes the .

Once the is formed, initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation.