RNA Polymerase II Complex
The detailed description of eukaryotic transcription was described following the determination of the structure of the twelve-subunit RNA polymerase. Subunits Rpb1 and Rpb2 make up the bulk of the complex, and the fold of these two regions forms an active core. Rpb3, 10, 11, and 12 are involved in assembly of the complex. Mobile domains of RNA polymerase form a “clamp” that shifts positions during transcription. The upper jaw of the clamp is comprised of regions of Rpb1, 2, and 9 (the jaw-lobe). Rpb1, 5, and 6 form what is known as the lower jaw. The movement of the clamp region of RNA polymerase is related to the rotation of the template DNA as it moves through the complex (Cramer, et al, 2001). Rpb4 and Rpb7 form a dimer and are the two subunits that are not included within the RNA polymerase holoenzyme. This dimer plays a role in the initiation of transcription. It interacts with both TFIIB and the mediator complex to stabilize the initiation complex (Bushnell, et al, 2003). Rpb7 also interacts with the clamp region of the holoenzyme to constrain its movement (Armache, et al, 2003).
Other key regions in the polymerase include 5 “switch” regions that connect the clamp to the core enzyme and a linker that extends from the clamp to the C terminal repeat domain (CTD) located at the end of Rpb1 (Armache, et al, 2003).
Initiation
The process of transcription begins with the assembly of an initiation complex, which consists of the 12-subunit RNA polymerase along with 5 transcription factors: TFII-B, -D, -E, -F, and -H (Armache, et al, 2003). TFIIB and TFIID are the key transcription factors that are required for the recognition of the promoter region and binding of RNA polymerase to the appropriate start site. TFIID binds to the TATA region of the DNA, about 25 base pairs upstream of the start site. The DNA is bent around the C terminus of TFIIB, and the N terminus of TFIIB interacts with RNA polymerase. This interaction between RNA polymerase and TFIIB is an essential event for the beginning of the transcription process. TFIIB is able to bind both the TATA box and RNA polymerase, and this binding brings DNA into a location where a straight path into the active center is available for the DNA. A distance of 25 to 30 base pairs from the site of TATA binding to the active site properly aligns RNA polymerase to begin transcription at the +1 site. (Bushnell, et al, 2004) A “finger” region of TFIIB also interacts with the DNA strand at several base pairs around the transcription start site. Residues 62-66, 69 and 74 interact with the -8 to -6 and -2 to +1 regions of the DNA strand. This interaction shows a method for start site recognition when a TATA box is not present at the -25 region.
Three other key transcription factors, TFIIF, TFIIE, and TFIIH, play different roles in the formation of the initiation complex. TFIIF interacts with the nontemplate DNA strand in its single stranded state. TFIIE is responsible for coordinating the location of TFIIH to a region downstream of the transcription site (about +25). Transcription factor TFIIH is involved with the process of “melting” the double stranded DNA to form a transcription bubble. TFIIH has an ATP-dependent helicase activity, and it will create a transcription bubble from approximately halfway to the TATA box (-25) to just past the transcription start site (+4) (Gnatt, et al, 2001). The formation of single stranded DNA allows the template strand to bend and enter the active site cleft to begin the RNA transcript. The single stranded DNA of the bubble is maintained by three loops that extend from the clamp. (Cramer, et al, 2001)
The entry of the DNA strand into RNA polymerase is facilitated by the swinging motion of the clamp region of the complex. In the second isolated form of RNA polymerase, the clamp has moved to create an opening for DNA entrance. The clamp is formed by both the amino and carboxyl termini of Rpb1 and by the carboxyl terminus of Rpb2. Three zinc ions are associated with the clamp region. There are five switches that connect the clamp to Rpb6, and these flexible regions allow for the movement of the clamp. The first three switches also interact with the RNA-DNA strand and facilitate the closure of the clamp after the template DNA has entered RNA polymerase (Cramer, et al, 2001). Also important for this process are the two subunits Rpb4 and Rpb7, which form the heterodimer associated with the clamp. The open state of RNA polymerase is not bound to these subunits. After promoter DNA has entered, the binding of the dimer to the clamp facilitates the closing of the clamp. The enzyme is held in the closed state by the restraining action of Rpb7, and this state persists throughout transcription (Armache, et al, 2003).
A mediator complex is the final required piece of the initiation complex. The mediator is large (1003 kD) and contains 20 subunits, and is responsible for relaying regulatory information to RNA polymerase. The mediator complex interacts with enhancers, or upstream activating elements, that are outside of the core promoter region for a gene. These enhancers regions may be hundreds of base pairs away from the transcription start site, and a mediator may interact with an enhancer without the presence of RNA polymerase initiation complex. (Kuras, et al, 2003) The mediator has three separate regions that associate with RNA polymerase. The main interactions take place on Rpb3 and Rpb11. The mediator increases the phosphorylation of the C terminal domain of Rpb1, which is necessary for transcriptional activation. (Davis, et al, 2002)
Elongation
Once RNA polymerase and all the necessary transcription factors have associated around the gene to be transcribed, the process of elongation begins. The major stages of elongation are: the unwinding and rewinding of double stranded DNA to form a transcription bubble, selection of a matched base pair nucleotide to the template strand, phosphodiester bond formation, and movement of the RNA and DNA strands through the RNA polymerase complex.
The process of unwinding the DNA double helix begins at the nucleotide position +5, where the site of ribonucleotide addition is denoted +1. At this point, the DNA template strand can be seen as single stranded, and is associated with Rpb1 in front of the active site cleft. Lysine 1109 and asparagines 1110 are the key residues on Rpb1 that interact with the single stranded DNA strand below the active center cleft. These interactions help form and maintain the transcription bubble downstream of the nucleotide addition site.
Once a single stranded DNA template is obtained, RNA polymerase is able to match a ribonucleotide to the template strand to form the growing RNA strand. The template strand nucleotide that is to base pair to the incoming nucleotide is in the position denoted +1, which is at the active site of the enzyme just above the base of the cleft (Gnatt, et al, 2001). The entrance and pairing of NTPs occurs at two overlapping sites, termed sites A and E. Two Mg ions are required for this process. One is associated with the α-phosphate of the incoming NTP and with residues 481, 483, and 485 (all aspartate) of Rpb1. The second ion is associated with the β and γ phosphates of the incoming nucleotide and with aspartate 481, aspartate 483, and aspartate 836 of Rpb2. Nucleotides enter through the E site and rotate up into the A site. If the match is incorrect, the nucleotide will not be able to hydrogen bond to the template strand, and will not assume the proper position within the active site. If the new nucleotide is a correct base pair match, it will be able to assume the proper position to continue with bond formation. Now in the A site, the phosphate of the NTP is exposed for a nucleophilic attack by an OH group that is required for the formation of phosphodiester bonds. The first metal ion plays an important role in this alignment, as it coordinates the arrangement of the 3’ OH of the nucleotide that is already attached to the growing strand and the α-phosphate of the incoming nucleotide in the A site. Once in this position, the two metal ions act to stabilize a transition state while the phosphodiester bond is being formed (Westover, et al, 2004).
The formation of a phophodiester bond between the 3’ OH of the RNA strand and the α-phosphate of the entering nucleotide completes one step in elongation. The DNA and RNA strands must then be translocated to make the A and E sites available for the entry of the next nucleotide. This translocation is accomplished by the bending of the helix bridge that spans the cleft between Rpb1 and Rpb2. The helix is straight in the standard conformation and a bend in the helix allows translocation of the nucleic acid strands (Cramer, et al, 2001).
The elongation of the RNA strand initially results in a DNA-RNA hybrid that is approximately 8 nucleotides in length. The RNA strand must be separated from the template DNA so the double stranded DNA helix can reform and the RNA strand may leave the nucleus to be translated. The two strands begin to separate at position -9 upstream of the currently open nucleotide. There are three protein loops from the RNA polymerase that appear to play an important role in the separation process. These loops are Rpb1 246-264 (the lid), Rpb1 310-324 (the rudder), and Rpb2 461-480 (the fork loop). The lid has the first important role as it wedges between the two strands beginning at the -9 position and then maintains the separation at position -10. The key residue in this process is the phenylalanine at position 252. The rudder is involved in maintaining the stability of the single stranded DNA at positions -9 and -10. Serine 318 and arginine 320 interact directly with the sugar-phosphate backbone of the unwound DNA. Instead of aiding in the unwinding, the fork loop region actually interacts with the DNA-RNA hybrid prior to the split. It is associated with positions -5 through -7 using lysine 471 and arginine 476. This interaction helps maintain an 8 base pair hybrid within RNA polymerase. There are also important interactions between these three regions of the polymerase (Westover, et al, 2004).
Termination
After the elongation phase has created an mRNA strand that contains the full gene that is required for protein translation, the mRNA must be removed from RNA polymerase. The process of termination is signaled by specific sequences of DNA found at the end of the transcribed gene. The RNA transcript exits RNA polymerase at a groove located near the C terminus of Rpb1. The exiting DNA is cleaved by TFIIS and RNA polymerase. TFIIS enhances the nuclease activity of RNA polymerase. A central domain (II) and a C-terminal domain (III) of TFIIS are required for the association to RNA polymerase, and domain III is responsible for the enhancement of nuclease activity. Domain III inserts itself into the exit pore of RNA polymerase and approaches the active site where the growing RNA chain is being formed. This active site is also the site of cleavage of the RNA. Aspartate and glutamate are two key acidic residues found on TFIIS that are required for the cleavage of RNA. The acidic residues interact with the backbone of the RNA strand and locate the appropriate end of the RNA strand. The release of the mRNA strand is coupled to the post-transcriptional modification of the RNA. A polyA tail is added to the 3’ end and a methyl cap is added to the 5’ end. RNA transcription is completed at this point, and mRNA proceeds outside of the nucleus to be translated (Kettenberger, et al, 2003).