We now have a general understanding of the main components involved the initiation of eukaryotic transcription. We will now shift gears and discuss how these components interact with one another by exploring the detailed mechanism of how this process works. Initiation involves the binding of RNAPII to the DNA, the unwinding of the DNA double helix, and the synthesis of the first few mRNA nucleotides.
RNAPII architecture and DNA binding
RNAPII’s 3-dimensional structure is mainly comprised of two large subunits, Rpb1 and Rpb2, which are surrounded by the other smaller subunits (11). At their interface, Rpb1 and Rpb2 form a cleft (View 2) in which the DNA double helix sits during transcription. The cleft also hosts the active site to which DNA binds to. This active site contains magnesium ions necessary for RNAPII catalysis. Both subunits contain extensions that form bridges over the cleft at sites remote from the active site. Both subunits are also held together by other smaller subunits, Rpb3, Rpb10, Rpb11, and Rpb12, which are located at one end of the cleft.
At the opposite end of the cleft, both the protein domains of Rpb5 and the protein domains of Rpb1 and Rpb9 form structures known as the “jaws” of the enzyme (View 3). These jaws both display mobility and use van der Waals interactions to grip onto the incoming DNA as it enters the cleft.
Another structure, referred to as the “clamp”, can be located on the side of the cleft that mainly contains Rpb1. This structure is comprised of certain regions of Rpb1, Rpb2, and Rpb6. The clamp serves to hold the DNA within the cleft and acts like a hinge on a door. It has one end that is fixed and one end that forms a bridge over the active site. View 4 is a top view of the enzyme in which DNA enters the bottom of the page and moves up the groove towards the top of the page. The red areas represent the clamp region which can close over the DNA-binding groove. Due to its function, the clamp is also thought to maintain the stability of the transcribing complex.
DNA unwinding and mRNA synthesis
The unwinding of the DNA double helix occurs three residues from the active site and forms a transcription “bubble.” This gives RNAPII access to read one of the strands (the template strand) and add complementary base pairs to synthesize mRNA in the 5’ to 3’ direction. The leading portion of this newly formed mRNA strand remains bound to the template strand of DNA and makes up what is called the DNA-RNA hybrid helix. This hybrid helix is about 9-12 base pairs in length and positions its newly formed 3’ end of mRNA at RNAPII’s active site. As new mRNA is synthesized, a region of the clamp displaces the old mRNA (located at the 5’ end) off the hybrid helix.
The bottom or floor of the cleft contains two large holes known as pore 1 and pore 2, which are located directly below the active site and the incoming (downstream) DNA respectively. These two pores meet at the opening of a funnel that leads to the outside of the enzyme (View 5). Because the downstream DNA and DNA-RNA hybrid helix block access to the top of RNAPII, nucleotides, the building blocks of mRNA, must enter through the funnel and pore 1 in order to reach the active site.
At the active site, two magnesium ions, termed metal A and B, coordinate phosphodiester bond formation (12). The substrates used for mRNA synthesis come in a precursor form, nucleotide triphosphates, which require the removal of the β and γ phosphate groups. During phosphodiester bond formation, metal A coordinates the 3’ end of the mRNA strand and the α phosphate group of the substrate. Metal B coordinates all three phosphate groups of the substrate. These two metals allow for the transfer of the nucleotide monophosphate onto the growing mRNA strand.
When the incoming DNA enters the cleft, it cannot move in a straight path to the other side of the enzyme (11). Instead, a protein region that forms a “wall-like” structure forces both the incoming DNA and the DNA-RNA hybrid helix at an upward angle. Therefore, when the 5’ end of mRNA is displaced off of the hybrid helix, it must move downwards through a channel known as groove 1. This groove starts at the base of the clamp and leads to outside of the enzyme. View 6 shows the back of RNAPII with the protein wall colored blue. Groove 1 (not colored) is directly below this wall.
When RNAPII begins transcribing the ORF at the start site, it is unable to move down the DNA to produce a long, continuous mRNA sequence (transcript). Instead, the enzyme stays in one place and produces several abortive transcripts, which are short copies of the first 2-9 nucleotides of the ORF (14). This initial process serves as several ‘test runs’ for RNAPII until it is ready to begin moving down the DNA.