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Prokaryotic vs. Eukaryotic

Eukaryotic Transcription

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Eukaryotic Transcription

Initiation:

The TATA box-binding protein of TFIID bends the TATA box DNA allowing the TFIIBC(C-terminal) binds sequences up and downstream of the TATA box (3).  This causes the DNA to curve and wrap around the RNA polymerase II.  The TFIIBN(N-terminal) binds to the dock domain, saddle, and active center cleft of RNA polymerase II.  When TFIIB binds the promoter DNA is directed toward and above the active center of RNA polymerase II, running across the face of the initiation complex, passing the Tfg2 subunit of TFIIF above the cleft passing TFIIE and TFIIH at the downstream end of the cleft (3).  The path of the DNA seems to be completely determined by interactions with transcription factors.  It is believed that the DNA does not interact with the RNA polymerase II before the DNA is separated into single strands (3).

TFIIE interacts with the RNA polymerase II jaws.  TFIIH, which is bound to TFIIE, has helicase activity and causes a quick promoter opening.  This open region of the non-template DNA is stabilized by the subunit Tfg2 of TFIIF.  The template strand is stabilized by the TFIIB finger and RNA polymerase in the active center cleft (3).  This allows the DNA to become more flexible because it is single stranded.  It is at this point that the DNA first bends and proceeds into the cleft.  The DNA then first interacts with RNA polymerase II in the active center and with what will become the transcription bubble.

The nucleoside triphosphates must be held in positions +1 and -1 for the synthesis of the first phosphodiester bond.  After the first few are added the nucleotides must still be held by protein-RNA interactions.  Sometimes these interactions are not sufficient to hold and the nucleotides are released.  This means the RNA polymerase must start again.  This is referred to as “abrortive cycling” (3). The TFIIB finger is only stabilizes a RNA/DNA hybrid for up to 9 base pairs after 9 base pairs the TFIIBN competes to occupy the RNA polymerase II saddle.  This is what is believed to account for the fact that TFIIB dissociates from the complex.  Before TFIIB dissociates TFIIH phosphorylates the CTD domain of Rpb1 (3).  This is the end of initiation.

Order of transcription factor binding:

  1. TFIID binds to the TATA box
  2. TFIIB binds to TFIID
  3. TFIIF and RNA polymerase II binds to TFIIB and TFIID
  4. TFIIH binds completing the initiation complex

Elongation:

Active Site:

The active site is described as area of nucleotide addition.  It contains two Mg2+ ions named A and B.  Metal A is bound is bound by the aspartates D481, D483, and D485 of Rpb1.  Metal B is coordinated by Rpb1 residues D481, D483, and Rpb2 residue D836 (10).

Transcription Bubble:

The downstream DNA lies in the cleft between the clamp and the subunit Rpb2.  The DNA contacts the “lower jaw” of subunit Rpb5 and then passes between the Rpb2 “lobe” and the Rpb1 “clamp head”.  The downstream portion of the transcription bubble starts where the DNA begins to unwind, which is approximately three to four nucleotides before the RNA/DNA hybrid.  The template strand proceeds along the bottom of the clamp over the helical “bridge”.  The +1 nucleotide is flipped compared to the +2 nucleotide by a left-handed twist of 90o(6).  This means that the +1 nucleotide points to the floor of the cleft and the active site.  Residues Ala832 and Thr831 position the +1 coding nucleotide and Tyr836 binds to nucleotide +2 to stabilize them.  The stabilization of the downstream portion of the transcription bubble can also be due to the binding to fork loop 2 of Rpb2.  The fork loop 1 of Rpb1 may also help stabilize the transcription bubble further upstream (6).

RNA Synthesis:

NTPs for transcription enter through the pore, which is found in Rpb1 in the floor of the cleft beneath the active site above the +1 site (6).  When the NTP enters the RNA polymerase II it brings with it metal B.  The NTP and B metal ion enter the E site (10).  If the NTP is the correct nucleotide the nucleotide is then rotated into the A site where the A metal coordinates the alpha phosphate of the NTP along with the aspartate residues 481, 483, and 485 from Rpb1.  The B metal is now coordinated by the beta and the gamma phosphates of the NTP as well as the aspartate residues 481 and 483 in Rpb1 and the asp residue 836 from Rpb2 (10). The nucleotide that was positioned at the +1 site has vander Walls interactions with Thr831 and Ala832 which are in the “bridge” that was described above on the Rpb1 subunit.  This helical bridge oscillates between a straight and bent conformation.  When the nucleotide is added at the +1 position would be straight.  When the bridge bends it translocates the nucleotide in the +1 position into the -1 position opening up the +1 site for another nucleotide to be added (6).  If the NTP is not the correct nucleotide it will stay in the E site and not rotate into the A site (10).

Separation of RNA from DNA and RNA Exit:

The separation of RNA from the DNA/RNA hybrid begins at position -8 with the residues after that being completely separated.  Three protein loops interact with the RNA/DNA hybrid to assist in separation: the “lid”, “rudder”, and “fork loop 1” (11).  The lid, which is composed of residues 246-264 from Rpb1, interacts with residues -8, -9, and -10 and acts as a wedge to break the RNA from the DNA.  It also maintains the separation and guides the RNA to the exit path.  The lid interacts with other proteins to form an arch  The plane of the aromatic side chain of Phe252  interacts with the hybrid and splits it.  The rudder, which is composed of residues 310-324 from the subunit Rpb1, prevents reassociation of the complex by interacting with the DNA at -9 and -10 (11).  This is accomplished by contact with the Ser318 and Arg320 at the 5’ phosphate at the -10 position.  Fork loop 1, which is composed of residues 461-480 of Rpb2, is believed to prevent unwinding of the hybrid past position -8.  The residues Lys471 and Arg476 appear to contact the RNA phosphates at positions -5, -6, and -7 (11).  These three loops lie near the saddle, which is underneath the arch, and between the clamp and wall.

The RNA exits through the so-called exit pore, which is the area beneath the arch.  After the arch the RNA follows one of two proposed exit paths.  In the first path the RNA follows a positively charged grove that runs around the base of the clamp and towards the Rpb7 subunit, which has the ability to bind single stranded RNA.  In the second path a positively charged groove that runs down the back of RNA polymerase II leads to the Rpb8 subunit, which could also interact with single stranded RNA.  The second path is favored because the RNA would have to take a sharp bend after passing beneath the lid if the first path was plausible (6).

Termination:

There is not much known about the process of termination in eukaryotes, but it is known that it is dependent upon poly(A) signals and downstream terminator sequences (7).  The details of this process are not very well known and are probably very variable.  What is known is that post-transcriptional modifications of the RNA occur at the same time as transcription.  The post-transcriptional modifications occur at the 5’ and the 3’ ends of the RNA molecule.  The 5’ end undergoes capping.  The first step is when the phosphoryl group is released by hydrolysis.  The diphosphate 5’ end then attacks the alpha phosphorus atom of GTP.  This forms a 5’-5’ triphosphate linkage that protects it from degradation from 5’ endonucleases.  This is referred to as the cap.  When the N-7 nitrogen of the terminal guanine is methylated it is referred to as cap 0.  When the other ribosomes are methylated it is referred to as cap 1 and cap 2.  The 3’ end contains a polyadenylated tail.  The RNA transcripts are cleaved by an endonuclease that recognizes the signal AAUAAA, which is part of the cleavage signal.  A poly(A) polymerase adds about 250 adenylate residues to the 3’ end of the transcript.  Another event in posttranscriptional processing is splicing.  In which the introns are removed from the RNA and the exons are then linked to form the mature mRNA.

The different subunits of RNA polymerase II. PDB 1I50

PDB 1TBP

The active site of RNA polymerase II with the various residues that coordinate the Mg2+ ions. PDB 1R9S

The clamp is shown in yellow, the pore in red, the cleft in green, and the jaw in blue. PDB 1R9S

The A site showing the coordination of UTP by the two Mg2+ ions. PDB 1R9S

The E site showing a mismatched base pair. PDB 1R9T

The rudder is shown in green, the lid is shown in red, and fork loop 1 is shown in blue. PDB 1SFO