User:Jordan Scott/Sandbox RNA polII

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 (mRNA). mRNA is a single stranded RNA molecule that is complementary to the template strand of DNA and matches the coding strand. In prokaryotes the transcript is ready for translation, but in eukaryotes post transcriptional modifications are required. The mRNA stand transports genetic information from DNA to the ribosome,where the code will be translated into an amino acid acid sequence to form proteins.

History

The Discovery and Isolation of RNA Polymerase by Jerard Hurwitz

Learning Objectives

Understand the structure of RNAP II Look at the processes of Initiation, Elongation, and Termination Gain a molecular and mechanistic view of these prcoesses

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.

Transcription

Transcription can largely be divided into three sections: initiation, elongation, and termination. In the process of initiation, RNAP II recruits several general transcription factors (GTFs) to bind to the promoter region of the DNA, and this eventually forms the preinitiation complex (PIC). The DNA enters RNAP II through the clamp, and then it is unwound, creating a transcription bubble. With the DNA unwound, the active site of RNAP II catalyzes the synthesis of the first few RNA bonds. Once the carboxy-terminal domain (CTD) becomes phosphorylated, the clamp undergoes a conformation change to effectively trap the DNA, and a few of the GTFs dissociate, which changes complex to the Elongator complex.

After initiation, the process of elongation begins with the entry of NTPs. These NTPs largely enter through the funnel, and this tends to be a slow process because the funnel is only 12 Å in diameter, meaning that only one NTP can go through at a time. Once they have passed through the funnel, the NTPs enter which contains an Mg ion that is bound by three aspartate residues at positions D739, D741, and D743. If the NTP is complementary to the DNA strand, it is loaded onto the insertion site next to the RNA and held in place by the bridge. While in this insertion site, the metal ion and aspartate residues catalyze the reaction that forms a phosphodiester bond between the 3’ end of the RNA and the 5’ end of the NTP. This step then leads into a translocation step. To make room for the next NTP, there is a Brownian ratchet mechanism in which the nearby trigger loop undergoes a conformation change that causes the bridge to move to the next transition site. During the bridge’s transition, the DNA-RNA hybrid is partially help in place by the -amanitin. Once the bridge is in the new initiation site, the trigger loop returns to its original conformation, allowing the process to begin again.

In the termination stage, the CTD becomes dephosphorylated, which acts as a signal to dissociate the elongation complex. Once this signal is received, the new mRNA is released from RNP II through the rudder. Before being released, its 3’ end is polyadenylated. The DNA is brought back together at the other end of the transcription bubble, returning it to its original double-stranded form. Finally, RNAP II and the remaining GTFs dissociate from the DNA.

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.

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 initiation factors(GIFs). The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is known as the preinitiation complex. This complex is necessary for an accurate initiation. 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:

Source: https://www.pnas.org/content/94/1/151.

1. is highly conserved among eukaryotes. It recognizes and binds the TATA box. This is facilitated by a subunit named the TATA-binding protein (TBP). This subunit also causes slight deformations in the helix which may be important for further binding of the PIC units.

2. is the second to join the PIC. It is thought to be responsible for stabilizing the TBP/DNA complex and insuring proper directionality . If the orientation is wring unfavorable interactions form. Mutagenesis studies also suggest that it works as a bridge between TFIID and pol II.

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

4. and are sequentially recruited which completes the .

5. is a co-activator that helps regulate PIC assembly. It serves as an enhancer and stabilizes the early complexes. It allows for inhibitory effect to be reduced and activators to be more effective.

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 and hlep with successive 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.