In Depth Eukaryotic Transcription

Initiation

In order for the TATA box binding promoter on TFIID to bind to the DNA, nucleosomes must be displaced from the area. The displacement of nucleosomes is achieved by chromatin-remodeling complexes. These complexes, disassemble nucleosomes by transferring the histone octamer of the nucleosome to another DNA molecule (1).

Next, the TATA box on the transcription factor TFIID recognizes and binds to the DNA. This binding causes the bending of DNA around the carboxy-terminus of the TFIIB domain for TFIIB association into the initiation complex. Next, TFIIF, bound to RNA polymerase II, enters into the initiation complex. The N-terminus domain of TFIIB interacts with the RNA polymerase in order to stabilize it into the initiation complex. The TFIIB contains a zinc finger domain that interacts with the polymerase dock domain. This zinc finger domain then passes across the saddle of the polymerase, between the clamp and wall regions down into the active site of the RNA polymerase II enzyme (3). Once the RNA polymerase II is bound to the initiation complex through the interactions with TFIIB, the downstream DNA is directed towards the active site of the enzyme. Next, TFIIE is associated to the initiation complex by interactions with the jaw domains of RNA polymerase II. The positioning puts TFIIE in place to contact the promoter DNA downstream of the transcription start site. Finally, TFIIE recruits TFIIH, which contains two ATPase/helicases and opens the DNA double-helix into single-stranded DNA at the start of the transcription +1 site. After the DNA at the transcription start site is unwound, Tfg2, a subunit of TFIIF, grabs a hold of the non-template strand and the template strand descends into the active site of the polymerase, where it interacts with the zinc finger domain of TFIIB. In order for the template strand to be recognized by the RNA polymerase II, the clamp region of the polymerase swings about 30 degrees in order to capture this strand (6). This conformational change is also accompanied by the ordering of five switch complexes, and three zinc ions in the clamp region. These changes direct the repositioning of this clamp region upon the recognition of the template strand. Once the clamp goes from an open position to a closed position, the RNA binding groove is opened up, and upon the opening and further binding of RNA, the clamp will stay in a closed formation. Finally, the enzyme is ready for the transcription of the first few ribonucleotides. The synthesis of the new ribonucleotides causes a clash between the newly formed RNA strand and the zinc finger from TFIIB. When this steric clash occurs, after about 8-9 ribonucleotides of the RNA chain have been produced there are two outcomes from the competition (3). The first is that TFIIB would prevent the transcript from growing past 10 residues. This would result in abortive transcription and the RNA polymerase II enzyme would leave the initiation complex with only an incomplete 10 ribonucleic acid strand produced. The second outcome is that the zinc finger domain on TFIIB would be displaced from the polymerase II enzyme, causing a dissociation of the TFIIB from the RNA polymerase II, which would result in the transition into the elongation phase from the initiation stage (1). Furthermore, the TFIIH has a kinase activity and phosphorylates the C-terminal domain of the Rpb1 subunit of RNA polymerase II. The phosphorylation coupled with the release of subunit TFIIB transitions the stage of transcription from initiation to elongation (3). For a visual view, check out the TFIIB commands in jmol below, specifically the menu.

Elongation

This downstream DNA contacts the jaw domain at Pro86 and Pro118 on Rpb5 (7). The DNA proceeds to pass between the lobe of the Rpb2, specifically by redisues 231-233 and the clamp head of the Rpb1. Finally, the template strand follows a path along the bottom of the clamp and over the bridge helix on its way to the active site (6). In order to maintain the transcription bubble during elongation, residues on Rpb1 and Rpb2 help to align the DNA correctly and separate the strands all the way to the +5 position. The Rpb1 residues of Lys1109 and Asn1110 interact with the +5 site of the nontemplate strand, fraying it from the template strand (9). Furthermore, the structure fork loop 2, from Rpb2 containing residues 500-510, assists in stabilizing the separated strands at the +3 and +4 positions.

Once at the active site, the nucleotide base +1 of the template strand is twisted 90 degrees and points downwards to the floor of the cleft so that the active site can identify the nucleotide (1 and 6). Since the DNA entering the active site is a single strand, it is able to accommodate for this twist easier. There are various residues on Rpb1, other than fork loop 2, and specifically at the cleft, which contains switches 1 and 2, and the bridge helix that stabilize the entry of the DNA into the active site. Switch 1, containing Rpb1 residues 1380-1410 stabilizes the DNA downstream of the active site. Specifically, Arg1386 and Glu1403 of this switch stabilize the +4 position. Also, Tyr836 on the bridge helix, Arg337 from switch 2, and Glu1403 of switch 1 stabilize the +2 position, downstream of the DNA. These residues are critical in order to stabilize the 90 degree twist from position +2 to +1. Many structures and loops from Rpb1 and Rpb2 stabilize the +1 region of the active site. Switch 2, which includes residues 331-338 of Rpb1 interacts with the +1 DNA base, as well as a loop containing residues 444-450, and finally, the bridge helix, containing residues 810-846 on Rpb1, stabilizes the +1 site (5). The two crucial residues at the bridge helix that interact with the nucleotides at the +1 position through Van der Waal interactions are Thr831 and Ala832. Thr831 functions to stabilize both the incoming RNA base and the DNA base at the +1 site, where as Ala832 primarily functions to stabilize the 90 degree twisted region between the +1 and +2 site on the DNA. A loop containing residues 480-486 also stabilizes the incoming NTP. The active site of this enzyme contains two magnesium ions that specifically interact with the incoming NTP. The first magnesium ion is bound to aspartates 481, 483, and 485 of Rpb1 and always remains in the enzyme (5). This magnesium ion functions to associate the NTP for the formation of the phosphodiester bond in order for it to be added to the RNA strand. To see an up-close view of the active site, refer to the active site jmol button above.

Next, an NTP molecule enters through the funnel and pore on Rpb1 coupled with the entry of a second magnesium ion (6). The second magnesium ion is shown to be bound 5.8 Angstroms away from the first magnesium ion. This second magnesium ion is located near three residues: Asp481 on Rpb1, and Glu836 and Asp837 on Rpb2 (5). The NTP molecule initially binds to an E site, and is then rotated around the second magnesium ion, which stabilizes the beta and gamma phosphates on the NTP molecule, into the A site. This rotation is thought to play a role in discrimination between rNTPs and dNTPs by Asn479 on Rpb1 (9). Once the NTP is flipped to the A site, the bridge helix holds the NTP in place. In the A site, the NTP is positioned adjacent from the template DNA strand with the alpha phosphate group in position for an in-line nucleophilic attack by the OH group of the 3’ end of the RNA molecule (9). After the nucleophilic attack has occurred, a new phosphodiester bond is made. Then, the pyrophosphate and the second magnesium ion exit the RNA polymerase II transcribing structure. Next, a translocation event occurs, where the newly synthesize RNA base and the template DNA base are moved and the next base on the template strand is twisted 90 degrees. This translocation event causes the A site to be vacant, in order for the next NTP to enter. The vacant A site represents a post-tranlocation state (1). The bending of the bridge helix on Rpb1 brings on this translocation event.

Furthermore, proofreading by the significance of hydrogen bonding and indirect discrimination from positions -1 to -5 occurs. This takes place where the RNA is associated with the DNA, and is used to make sure that the RNA is correctly associated with the DNA of the template strand. This proofreading occurs by the removal of the RNA by cleavage at the active site through the mechanism of TFIIS (4). Before the RNA can be cleaved, the RNA must be backtracked in order to get to the mismatched site, and the RNA is cleaved at this site. This transcription factor TFIIS contains an extended beta-hairpin which extends through the funnel and pore 1, which is the pathway of the incoming NTPs, and contains two residues that go near the active site of the RNA polymerase II, which are involved in the RNA cleavage mechanism (7).

Along with the proofreading from positions -1 to -5, there are also many structures and loops that help stabilize the DNA-RNA helix in order to maintain contact with the protein. One major structure is switch 3, containing residues 1120-1133 on Rpb2. Met1133, Arg1129, Leu1128, Gly1121, and Gly1123 stabilize the phosphodiester bonds on DNA between the residues +1 and -1, -1 and -2, -2 and -3, -3 and -4, and -4 and -5, respectively. Furthermore, Arg1122 stabilizes the DNA base at the -4 position. The wall from Rpb1 comes in contact with the DNA base +5 to stabilize it, as well as residues Met792 and Thr791 from Rpb2. To maintain polymerase contact with the RNA strand, Arg446 from a loop on Rpb1 stabilizes the -1 base. Also, residues Lys987 and Lys 979 from Rpb2 stabilize the -2 RNA base. Furthermore, Asn776, His1097 and a loop containing residues 528-532 on Rpb2 function to stabilize the -3 base of RNA (5). To see all of these residues relative to the DNA and RNA, refer to the DNA and RNA Stabilization button above in the jmol images.

After specific checks and interactions with DNA and the RNA polymerase II, the DNA and RNA are dissociated from each other. Three main regions are involved with the dissociation of the DNA from the RNA. From positions –5 to -7, fork loop 1, located on Rpb2, interacts with these phosphate groups, specifically by residues Lys471 and Arg476 (8). The fork loop 1 serves to prevent unwinding of the DNA-RNA strand up to the upstream position -8. The rudder interacts with the DNA at positions -9, -10, and -11 and the RNA at positions -7 and -8. The function of the rudder is to prevent DNA reassociation with the RNA and to stabilize the single-stranded DNA beyond the hybrid region. Specifically, residues Ser318 and Arg320 from Rpb1 come in contact with the sugar and 5’ phosphate of the -10 DNA base (8). The lid serves to split the DNA and RNA strands apart by wedging between the double-stranded helix. It interacts with bases -8, -9, and -10 of the RNA, and specifically splits the DNA-RNA strand at position -10. The tip of the lid domain consists of a Phe252 residue, where the aromatic side chain comes in contact with the DNA base and splits the DNA-RNA apart. Furthermore, Phe264 plays a similar role in this mechanism and comes in contact with either the -10 or -11 positions (8). Along with these three integral regions, a loop containing residues 206-208 of Rpb1 and the finger region, containing residues 440-464 function to stabilize the downstream DNA bases -7 and -8. Once the strands are dissociated from one another, the lid forms a barrier to keep the strands separated and to guide the RNA along an exit pathway. The lid forms an arch over the saddle so that the RNA exits beneath the arch and the DNA exits above the arch, preventing the possibility of reassociation. From this point, the RNA exits through a positively charged groove running down the back side of polymerase II (6). Also, the single stranded DNA exits and conjugates back with the non-template strand to form the end of the transcription bubble.

Termination

Once the DNA bases have been transcribed and the RNA chain is complete, termination occurs. In eukaryotic transcription, termination involves the poly-A signals and downstream terminator sequences. After the poly-A signals have been transcribed, a pause element pauses the transcription process. This pause may occur after as many as 200 ribonucleotides have been transcribed after the poly-A signal. After the pause occurs, the endonuclease cleaves the RNA after this poly-A signal of AAUAAA. After the RNA has been cleaved, it undergoes many post-transcriptional modifications. These modifications only occur in the pre-mRNA products, which are transcribed by RNA polymerase II. One of these modifications is the addition of a 5’ cap. This capping process actually takes place immediately after the beginning of transcription. The process for the addition of the cap is very unusual. First, the phosphoryl group of the 5’ terminal nucleotide, usually an A or G, is released by hydrolysis. The resulting diphosphate on this terminal base proceeds to attack the alpha-phosphorus on the second nucleotide, GTP, which forms a 5’-5’ triphosphate linkage. Furthermore, the N-7 nitrogen of the terminal base is then methylated by S-adenosylmethionine to complete the formation of the cap. These caps contrbute to the stability of the mRNA by preventing degradation of nucleotides from the 5’ end from phosphates or nucleases. Moreover, the caps enhance the translation of the mRNA. A second post-transcriptional modification of the pre-mRNA product is the addition of a poly-A tail. A poly(A) polymerase adds around 250 adenylate bases to the 3’ end by the use of ATP. This poly-A tail is thought to enhance the efficiency of translation of this mRNA and it is thought to provide stability and extend the half-life of the mRNA product. RNA editing can also take place, which are catalyzed by deaminases. Finally, splicing occurs in the pre-mRNA transcript by two transesterification reactions in order to complete the pre-mRNA to mRNA modifications. Small nuclear RNAs in spliceosomes catalyze these two reactions. Splicing results in the excision of the introns and alternative splicing forms different mRNA products from the same gene, which encode different functional proteins.