Initiation
The first step in the RNA synthesis process is called initiation. RNAP II (Show Subunits) from yeast and humans typically has 10 to 12 subunits, which are capable of synthesizing RNA but require the use of transcription factors that are needed for specific promoter binding [3]. Transcription initiation is a very sophisticated biochemical process that is overseen by interactions between many units. These units include the RNAP II, multiple accessory transcription factors, ATP-dATP cofactor, and DNA [1]. There are as many as six general transcription factors which will assemble with RNAP II and allow for promoter recognition and the melting of the DNA [2]. To start the process of initiation, one or more accessory factors will bind to the promoter sequences and form a stable intermediate in transcription initiation [1]. RNAP II from humans, which consists of 12 subunits (Rpb1-Rpb12) and has a mass of >0.5 MD [2], will recognize the initial complex and with the assistance of additional factors, it will bind to this complex and form the preinitiation complex [1]. Using the ATP-dATP cofactor, the preinitiation complex will be converted to an activated complex which once introduced to RNAs can initiate rapid RNA synthesis [1].
There are six general transcription factors (GTFs) that are needed for specific promoter binding which are TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH [3]. The preinitiation complex of the GTFs and RNAPII are assembled in a stepwise fashion [3]. These GTFs are assembled in a specific order starting with TFIID, followed by TFIIA, TFIIB, RNAPII TFIIF, TFIIE and finally TFIIH [3]. This assembled apparatus will continue through several steps to form elongating complexes. The complex will melt the DNA so that about 12-15 bp of DNA are in the single strand form. The polymerase will then form a couple phosphodiester bonds The complex will usually go through something known as abortive initiation which is caused by the polymerase repeatedly initiating transcription and that will cause the release of small RNA strands [3]. This will usually occur until the polymerase can form a longer RNA strand and it will move past the abortive initiation stage. The complex will then stop 25-30 bp before the promoter sequence which will allow the complex to make a transition to allow for promoter escape [3].
Elongation
In order to form a transcript, the RNA initiation complex must change after promoter clearance and to allow the elongation to occur. An important change that does occur involves the carboxy-terminal-repeat domain (CTD). The CTD is closely related to the phosphorylation state of RNAPII [3]. RNAPII found in initiation, usually does not have a phosphorylated CTD while RNAPII that is elongating usually has heavily phosphorylated CTD [3]. It appears that if the phosphorylation of the CTD actually causes the RNAPII factor to switch cofactors. RNA processing factors will become tightly associated with HNAPII that has a phosphorylated CTD [3].
The major factor in transcription elongation has to do with the two largest subunits of RNAPII, Rpb1 and Rpb2 (DNA Binding Site). These subunits lie at the center and on either side of the nucleic acid binding cleft and also include many smaller subunits around the region [4]. These two subunits will interact with each other around the subunit and also have an α-helix (known as the bridge helix) that will bridge the two subunits [4]. The movement and position of this bridge plays an important role by binding both the second and third unpaired DNA bases and it also binds to the coding base paired with the first residue of the RNA [4].
Active Site Structure
DNA is suggested to enter a positively charged cleft (Cleft) that runs between Rpb1 and Rpb2 which leads to the active site (Rpb1 Loop) which is marked by a Mg2+ ion[2], [5]. Since these are mobile elements, they have been given the term “jaws” [2]. Past the active site, where DNA strands are separated, the DNA runs into a protein “wall” [2]. The DNA-RNA hybrid that ends up leaving the active site has to actually pass the wall at almost a right angle. Since this would cause quite a bit of torsion, the DNA-RNA hybrid are held in place by a large “clamp” that will settle over the active site (Show Clamp) [2], [5]. There is also a hole (Pore 1), known as pore 1, on the floor of this cleft that allows the addition of NTPs and is also an exit for RNA during RNAPII retrograde motion [2].
There are two forms of RNAPII which are called form 1 and form 2. Three modules will change positions relative to the core polymerase which will cause the changing between form 1 and 2[2]. These three modules lie along the cleft but before the active site and form a structure known as the upper jaw and lobe [2]. The remaining structures are known as the shelf, lower jaw, assembly domain, the foot and cleft region with the final module being the clamp which has been identified as the mobile element in RNAPII [2]. The changes between form 1 and form 2 are just small rotations of the upper jaw and lobe which cause individual residue movements of up to 4Å, with a larger swinging motion of the clamp which results in a 14Å movement [2].
Once promoter DNA has entered the cleft, the clamp will swing over the cleft which has three effects; it will open the cleft further to allow DNA to move through the active site easier, it will trap the template and transcript and will also separate DNA and RNA strands at the upstream end of the transcription bubble[2], [5]. The clamp is stabilized by three Zn2+ ions and mutations that cause these ions to be missing will produce lethal phenotypes [2]. The clamp itself is a rigid unit but there are five switch regions that will go through conformational changes and transitions. Switches 1, 2, and 3 will become highly ordered and will contact the DNA-RNA hybrid in the active center [2], [5].
There is a loop near the active site that is involved in the maintenance of the edge of the transcription bubble [2], [5]. This is very important to keep the bubble open and at the same time allowed for interaction in the active site and the formation of the DNA-RNA hybrid (DNA-RNA Hybrid). Normally, the DNA strand is disordered but the +4 position (three nucleotides before the beginning of the DNA-RNA hybrid) has an ordered nucleotide [5]. This will allow the template strand to move under the clamp and over the bridge helix. The base at nucleotide +1 points towards the floor of the cleft to allow for readout at the active site while the next nucleotide is in the opposite direction. This orientation is obtained by the +1 base interacting with switches 1 and 2 as well as binding to the bridge helix [5]. This position will be important to the addition of NTPs on the RNA strand.
Interaction of metals
There are two Mg2+ ions that are in the active site (Mg ion) and both play a crucial role in transcription. Metal A is in the active site of RNAPII and is in a prime position for the binding of a phosphate group between the +1 and -1 positions on the developing RNA strand[2], [5]. The ribonucleotide at position +1 lies at the entrance of pore 1. This close association shows strong support for NTPs entering through the pore during RNA synthesis and RNA protrusion through the pore during back tracking and this leaves no other way for NTPs to enter the active site unless there are major conformational changes[2], [5]. Large changes are unlikely as that would most likely cause disruption of nucleotide-protein interactions. Metal B is located further down near the pore and helps to coordinate all three phosphate groups of the triphosphate. Metal B has been proposed to come in with the NTP and lining up the triphosphate so that a phosphodiester bond can be formed [2], [5].
Westover et al. [7] showed that the use of two ions in the active center is crucial to the transcription process. They also showed that there are two NTP binding sites on RNAPII which are known as A and E sites. Metal A, as stated before, sits in the active site known as site A, while metal B occupies site E [7]. Site E sits in a spot where NTPs that are entering RNAPII will go to E first before moving to A. The Mg ion (metal A) is permanently in the A site while metal B will enter the E site with the NTP. That of course means that metal B will bind to the NTP prior to its entry into the E site. These sites overlap so that they cannot simultaneously bind an NTP [7]. The significance of this newly discovered E site is that incoming NTPs will bind there first. This will allow the NTP to rotate around metal B and sample the base pairing in the A site [7]. This allows the NTP to test to see if it is the correct nucleic acid that is supposed to be added next. This allows RNAPII to backtrack and release the incorrect NTP and bind the correct one [7].
Base Pair Stepping
Until recently, there have been two proposed models as to translocation during transcriptional elongation. The first suggests that a power stroke is tightly coupled to a pyrophosphate release which drives motion. The second hypothesizes that the reversible diffusion of enzyme along the DNA template is rectified by the binding of the incoming NTP which causes a Brownian ratchet mechanism [6]. What Abbondanzier et al. discovered was that the translocation distance is about 3.4 Å which would correspond to about 1 bp which is what would be needed for accurate transcription. They also showed that increasing pyrophosphate concentration has no effect on the elongation speed therefore suggesting that a power stroke would not be what is causing translocation [6]. Coupling this with the fact that a power stroke will cause a larger movement than the 3.4 Å to move it one base also points towards the ratchet mechanism. They propose that this mechanism uses a secondary NTP binding site which when the next nucleotide binds to the site, which will cause translocation to the next nucleotide [6].
RNA Exit
When the transcript reaches about 10 residues in length, the RNA must separate from the DNA-RNA hybrid and move to an exit channel which will protect the strand from nuclease attack for about six more residues [5]. There are three loops known as the rudder, lid and zipper which have roles in the dissociation of the hybrid strand, RNA exit and maintenance of the transcription bubble [5]. The separated RNA will move out of RNAPII through exit groove 1[2], [5]. The claim that groove 1 really is the exit groove is strengthened by the fact that groove 1 is near the CTD, capping and other RNA processing enzymes [2]. This will allow for the exiting RNA to be capped, poly-adenylated, and spliced as it leaves RNAPII.
Termination
Termination is defined as the permanent discontinuation of RNA synthesis along with RNAP-DNA dissociation. It has been determined that termination elements located in the 3’ region of genes are responsible for coordinating temporal and spatial interactions between the poly(A) site nascent transcript and the DNA template. Therefore, that means the promoter, poly(A) site, and the termination element of a gene must interact to bring about the most effective termination [8].
[1] Conaway, J.W.; Conaway, R.C. An RNA Polymerase II Transcription Factor Shares Functional Properties with Escherichia coli σ70. Science. 22 June 1990. 248, 1550-1553.
[2] Cramer, P.; Bushnell, D.A.; Kornberg, R.D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 8 June 2001. 292, 1863-1876.
[3] Lee, T.I.; Young, R.A. Transcription of Eukaryotic Protein-Coding Genes. Annu. Rev. Genet. 2000. 34, 77-137.
[4] Bushnell, D.A.; Cramer, P.; Kornberg, R.D. Structural basis of transcription: α-Amanitin-RNA polymerase II cocrystal at 2.8 Å resolution. PNAS. 5 February 2002. 99, 1218-1222.
[5] Gnatt, A.L; Cramer, P.; Fu, J.; Bushnell, D.A.; Kornberg, R.D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 8 June 2001. 292, 1876-1882.
[6] Abbondanzier, E.A.; Greenleaf, W.J.; Shaevitz, J.W.; Landick, R.; Block, S.M. Direct observation of base-pair stepping by RNA polymerase. Nature. 24 November 2005. 438, 460-465.
[7] Westover, K.D.; Bushnell, D.A.; Kornberg, R.D. Structural Basis of Transcription Nucleotide Selection by Rotation in the RNA Polymerase II Active Center. Cell. 12 November 2004. 119, 481-489.
[8] Gromak, N.; West, S.; Proudfoot, N.J. Pause Sites Promote Transcriptional Termination of Mammalian RNA Polymerase II. Molecular and Cellular Biology. May 2006. 26, 3986-1996.