Elongation begins after the formation of the initiation complex and the first 3 nucleotide of the mRNA is constructed.
RNA synthesis/Active site: Mg2+ is an important ion in the active site of the RNA synthesis. There are 2 Mg2+ ions present. The first Mg2+ ion is bound to Rpb1 (amino acid D481, D483, and D485) and is referred to as metal A. The metal marked as A is responsible for binding to the phosphate group between the nucleotides at the 3’end of the RNA and the adjacent nucleotide to coordinate the 3’OH group. Due to the importance of metal A, it is consistently bound to the upper edge of the pore of the RNA synthesis active site. The second Mg2+ ion, Metal B, has the ability to bind to all 3 phosphates this allows Metal B to dissociate from the active site and leave along with the pyrophosphate after nucleotide addition. Metal B is located to Rpb1 at amino acid D481 and E836 and D837 of Rpb2 [5] which is located relatively close to metal A. Nucleoside triphosphates (dNTP’s) can enter through the pore in RNA synthesis and that RNA is extruded through the pore during backtracking.
The clamp: The role of the clamp is to hold the DNA and RNA together over the cleft and active center. The clamp however, displays a swinging motion that is thought to produce a openings of the cleft to permit the entry of promoter binding molecules for the initiation of transcription. The clamp becomes very useful in that, it is able to detect the DNA-RNA hybrid conformation and separate the DNA and RNA strands at the upstream end of the transcription bubble [5]. The formation of the clamp is due to the NTD region of Rpb1 and CTD regions of Rpb2. The clamp head (upper region) allows for the stability of the 3 Zn2+ ions. The clamp is further connected to different regions such as the cleft region of Rpb1, the anchoring region of Rpb2, and to a set of switches in the Rpb6 region. Overall the role of the clamp is for stability and anchoring the DNA and RNA in position.
The switches: There are 5 possible switches that can undergo conformational change. As mentioned in the clamp, switches 1,2,4, and 5 form the base of the clamp. In a free Pol II, switches 1 and 2 become poorly ordered (not well structure for elongation) and switch 3 is disordered. But during transcription the 3 switches become well ordered from binding to the DNA downstream [9]. Switches 1,2,and 3 are able to make contact with the DNA-RNA hybrid in the active center.
Translocation: The bridge helix from Rpb1 was thought to be the cause of movement in the DNA template. It was shown that the bridge helix was highly conserved in RNA polymerase in bacteria. The observed bents and straightening of the bridge helix was thought to mediate the movement by 3 to 4 Angstroms. Translocation at the +1 would straighten the bridge helix while; a translocation at the -1 site would present a bent bridge helix. This was thought to produce the movement in the production of RNA [9].
The movement of the DNA through Pol II has been described and studied. The DNA enters through a cleft down the middle of the enzyme passing between a pair of mobile elements that are “jaws-like”. The jaw is composed of 2 halves. The upper jaw is made of Rpb1, RPb9, and the lobe of Rpb2 regions. The shelf module contains the lower jaw (a domain of Rpb5), the assembly domain of Rpb5, Rpb6 and the foot and cleft regions of Rpb1 [5]. After passing the active site, a protein wall blocks the DNA path, however; the DNA is able to pass through Pol II in a straight path through a cleft from one side of the enzyme. The RNA-DNA hybrid is able to pass the wall through a 90o angle in the cleft. In this path DNA passes to the jaw domain of Rpb9 and fits into the concave surface of the Rpb2 lobe where is surrounded by switches 2, 3, the rudder, and the flap loop [7]. Refer to the figure below.
RNA exits: The 2 main grooves visible in Pol II are located at the base of the clamp (groove 1) and located alongside the wall (groove 2). When the transcript reaches 10 residues in length, the newly synthesized RNA must separate from the DNA-RNA hybrid and exit through a channel on the surface. Maintenance of the transcription bubble upstream requires the movement of RNA from the active site. The zipper and lid play a role in separating the template and the non-template DNA strands. The 5’ end of the RNA exits though groove 1 during RNA synthesis as Pol II moves forward. The 3’ end of the RNA is pushed into the enzyme during retrograde movement. The rudder lies beneath the RNA from the last hybrid base pair and is responsible for separating the RNA from DNA and maintaining the upstream end of the RNA-DNA hybrid.
Eukaryotic Termination