Now that you are familiar with the general overview of the transcription process I will take you into our current understanding of how transcription works at a detailed level. The DNA of eukaryotes is ordinarily non responsive to transcription because of its organizations in nucleosomes, which is a general gene repressor. This is where the DNA is wrapped around a histone octamer causing interference with many DNA transactions. This association helps assure genes are not expressed at inappropriate times in eukaryotes, except in genes whose transcription is brought about by specific positive regulatory mechanisms. So, in order to initiate transcriptional activation these nucleosomes must be removed from all most all promoters in the process of transcriptional activation (Boeger, et al, 2004). We currently believe that nucleosomes are removed by disassembly and not by sliding. The disassembly mechanism appears to involve chromatin-remodeling complexes, such as the 11-subunit SWI/SNF complex and the abundant and essential 15-subunit RSC complex. RSC has been shown to disassemble a nucleosome by transferring the histone octamer to another DNA molecule with the help of the histone octamer-acceptor protein Asf1 in yeast. Although, it is still unknown whether RSC or another chromatin-remodeling complex transfers histone octamers from the promoter to Asf1 in the process of transcriptional activation (Boeger, et al, 2004).
Initiation
After the nucleosomes have dissociated from transcriptionally active promoters, the RNA polymerase II transcription initiation complex is assembled on the naked DNA molecule at the promoter region. There are essentially three components of the initiation complex. These include the five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), the 12-subunit polymerase, and the 20-subunit Mediator, which is unique to eukaryotes (Bushnell, et al, 2003).
The RNA polymerase II enzyme contains 12 subunits. The two largest subunits (Rpb1 and Rpb2) fold together to form an active core. Rpb1 contains the C terminal repeat domain (CTD) (Armache, et al, 2003). Rpb3, 4, 7, 10, 11, and 12 are believed to be involved in assembly of the complex. A “clamp” region makes up the mobile portion of the polymerase which consists of Rpb1, 2, 5, 6, and 9. The movement of the clamp region of RNA polymerase has been linked to the associated of DNA and the recognition of DNA-RNA hybrid (Cramer, et al, 2001).
Our current understanding of how the initiation complex is assembled on DNA goes as follows. The two most important transcription factors are TFIID and TFIIB, which are responsible for recognizing the promoter and making sure RNA polymerase binds to appropriate start site. To accomplish this, the TATA box binding protein (TBP), subunit of TFIID, bends the TATA box DNA creating an open binding site for the binding of the C terminus of TFIIB and a curvature of the DNA that is appropriate for wrapping around pol II (Bushnell, et al, 2004). The C terminus of TFIIB then binds sequences upstream and downstream of the TATA box. The N terminus of TFIIB binds to the dock domain, saddle, and active center cleft of pol II. TFIIB determines the location of the transcription start site by C terminus TFIIB binding at the TATA box, and fine setting by the finger domain of N terminus TFIIB interacting with the template DNA single strand. The finger domain of TFIIB also interacts with the DNA strand. If a TATA box is not present at the -25 region then the start site could be recognized by the finger residues 62-66, 69 and 74 which interact with the -8 to -6 and -2 to +1 regions of the DNA strand.
The binding of TFIIB causes the promoter DNA to be directed toward and above the active center region of pol II bringing the transcription start site in the DNA close to the active center. The distance of 25 bp is important because it allows the proper alignment of RNA poly at the +1 start site.
The TFIIE, TFIIF, and TFIIH transcription factors all have their own roles in the formation of the initiation complex. Now that TFIIB and the promoter DNA are in the complex, TFIIE can bind which then recruits TFIIH to a position downstream of the transcription start site. TFIIH has an ATPase/helicase subunit which torques the DNA introducing negative superhelical tension in the region over the active center cleft and melts the DNA creating a transcription bubble halfway from the TATA box to the transcription start site +1 (Gnatt, et al, 2001). Once the DNA is in single stranded form TFIIF will then capture the non-template strand and the template strand is then able to enter the active site cleft. The melting allows the DNA template to bend by 90o so the template strand can enter the active site cleft of the polymerase.
The swinging motion of the clamp region of the complex facilitates the entry of the DNA into this cleft. The clamp fold seems to be formed by NH2- and COOH terminal regions of Rpb1 and the COOH-terminal regions of Rpd2 and is stabilized by three Zn2+ ions which have been shown to be essential for survival (Cramer, et al, 2004). The clamp is connected to the Rpb1, Rpb2, and Rpb6 regions by its base. The Rpb6 region contains 5 switches which undergo conformational changes in the transition to a transcribing complex, and three of the switches contact the DNA-RNA hybrid in the active center cause the clamp to close in the presence of DNA-RNA hybrid, which is key to the transcription process.
The Mediator makes up the final portion of the initiation complex and is made up of 20 subunits. The Mediator interacts with upstream promoting elements and enhancers that lie outside of the core promoter region of the gene. Although the exact mechanism of communication is unknown, it is clear that Mediator interacts extensively with Polymerase II stimulating transcription, as Mediator has been shown to bind Rpb1, 3, and 11 (Kornberg, et al, 2005). Mediator has also been shown to increase the phosphorylation of the CTD of Rpb1, which is essential in transcriptional activation (Davis, et al, 2002). The Mediator seems to be the key to understanding how multiple regulatory signals are processed and transmitted to RNA polymerase II and transcription regulation in eukaryotes (Kornberg, et al, 2005).
Elongation
After the assembly of the initiation complex, the process of elongation begins. Elongation can be divided into the following processes: the maintenance of the transcription bubble, selection of a matched base pair nucleotide followed by phosphodiester bond formation and translocation of the DNA-RNA hybrid helix during transcription.
The transcription bubble is maintained on the downstream edge of the transcription bubble by the binding of nucleotides +2, +3, and +4 and also the Rpb2 “fork loop” 2 (Gnatt, et al, 2001). Nucleotides +2, +2, and +4 are disordered in such a way that they are stacked like right-handed B-DNA. This unusual conformation results from the binding of the nucleotides to switches 1 and 2, as well as the bridge helix. Rpb2 fork loop 1 may have a role in maintaining the transcription bubble further upstream with help from the rudder and lid. Fork 1 and fork 2 seem to play an important role in separation of the DNA-RNA hydrid after translocation.
Nucleoside selection occurs when nucleotide triphosphates (NTPs) diffuse to the active center of RNA poly II through a funnel-shaped opening. The actual selection of nucleotides that flow through the active center is believed to occur at two overlapping sites, termed A and E. Nucleotides enter through the E site and rotate up into the A site. If an incorrect nucleotide is bound, then the phosphate required for the formation of phosphodiester bonds is exposed for nucleophilic attack by a hydroxyl group. If the base is a correct match, then a Mg ion plays an important role in aligning the 3’ OH of the nucleotide and the a-phosphate of the incoming nucleotide in the A site (Gnatt, et al, 2001). The two Mg ions help stabilize this transition state while the phophodiester bond is being formed (Westover, et al, 2004). One of the Mg ions associates with Rpb1 and the a-phosphate of the incoming nucleotide and the other Mg ion interacts with the b and g-phosphates of an incoming nucleotide and Rpb2. A correct nucleotide facilitates the formation of a phosphodiester bond between the 3’ hydroxyl of the RNA strand and the a-phosphate of the correct nucleotide. Translocation of the DNA-RNA hybrid helix involves the bridge helix which acts as a ratchet to make the A and E sites available for the next nucleotide and the cycle is repeated. The side chains of threonine-831 and alanine-832 contact the nucleotide base and actual translocation is accomplished by oscillation of the bridge helix between straight and bent states which move the DNA-RNA hybrid one nucleotide step (Boeger, et al, 2004). It is very important for the NTPs to be held in positions +1 and -1 until the synthesis of the first phosphodiester bond. After translocation to positions -1 and -2, the dinucleotide product must still be held by protein-RNA contacts, because base pairing is not sufficient for retention in the complex. After position -4 RNA is exposed with no direct protein contacts for stabilization except for the hydrogen bond at -5. This leads to abortive cycling which yields RNA transcripts of less than 10 residues. TFIIB has a loop that stretches into the active site of the polymerase where it competes with the newly formed DNA/RNA hybrid. If the loop wins, then it binds to DNA causes abortive initiation, but if the RNA chain wins then the loop of TFIIB is displaced along with the promoter DNA allowing RNA polymerase to escape from the promoter and start elongation.
After elongation has started it is very important that the RNA strand be separated from the template DNA so the RNA can leave and the double stranded DNA helix can reform. There are three loops that play key roles in RNA-DNA separation and they are the lid (Rpb1 246-264), rudder (Rpb1 310-324), and the fork loop 1 (Rpb2 461-480). The lid serves as a wedge between the RNA and DNA strands driving them apart by interacting with the residues -8, -9, and -10. Phe252, on Rpb1 residue splits the RNA-DNA base pair at position -10. The rudder interacts with the DNA at -9, -10, and possibly -11 preventing reassociation with the RNA. The Rpb1 residues Ser318 and Arg320 contact the sugar and 5’ phosphate at -10 on the DNA. Fork loop 1 may be responsible for prevention of hybrid unwinding past -8. Rpb2 residues Lys471 and Arg476 appear to contact the phosphates facilitating this process. The loops not only interact with the DNA and RNA, but they also interact with one another giving rise to a complex called “strand/loop network” (Westover, et al, 2004). The process of termination is signaled by a specific sequence of DNA at the ends of genes.
Termination
Elongation has now created a messenger RNA that contains the full gene carrying the information for protein synthesis. After termination has been stimulated the RNA transcript exits RNA polymerase near Rpb1. TFIIS stimulates RNA polymerase to cleave the exiting DNA. There are two domains (central domain II and C-terminal domain III) on TFIIS required for association with RNA polmerase. Domain III goes into the exit pore and two key acidic residues (aspartate and glutamate) facilitate the cleavage of RNA (Kettenberger, et al, 2003).
*The mRNA undergoes further post-transcriptional processing with the addition of a Poly-A tail, capping, and splicing of introns.