PROCARYOTIC VS. EUKARYOTIC TRANSCRIPTION

 

 

Bacterial and Eukaryotic polymerases, besides having similar function, carry much of the same structural motifs and similar binding domains. Bacteria only have a single polymerase to take care of each duty that RNA polymerases have. Where as Eukaryotes (Figure 1) have three separate polymerases, one according to each different type of RNA transcript that is synthesized.

 

 Considering both Bpol and pol II RNA polymerases produce a transcription product unit, or here mRNA, that extend from the promoter sequence to the termination sequence on the DNA transcript, and have similar subunits, further examination into the general activation of transcription process is appropriate. Remembering that the entire transcription process can be broken down into three parts, Initiation, Elongation and Termination, comparison of the two polymerases can be considered. Detailed examination of the pol II transcription process can be found in Section 3.

 

INITIATION Binding of the RNA polymerase and association of the initiation complex to promoter and enhancer regions on double-stranded DNA in order to begin synthesis of the RNA transcript. 

Focusing on the similarities and differences of bacterial RNA polymerase (Bpol) and most common polymerase II  (pol II) from eukaryotes and involvement in the synthesis of mRNA, we can get a better understanding of how each polymerase as similar as they may be, function according to their environment. Examination of each polymerase individually and then comparing them, conclusions on the similarities and differences in the sequence, structure, promoter binding domains and the actual promoter sequences that these remarkable proteins have with each other. Once we understand each polymerase and initiation complex individually this will lead to a better understanding of how the transcription process of each polymerase works.

Lets first look at the Bacterial RNA Polymerase (RNAP) complex (Model 1 at bottom), which I will call Bpol or RNAP. During initiation and formation of the transcription bubble the complex goes from a closed (RF_c) conformation through two intermediate stages I1 and I2 to an open (RF_o- forked junction) one. (1) This transcription protein can be looked at as comprised of a core element and a holoenzyme structure. The RNAP holoenzyme contains four subunits: alpha (A), beta (B), beta' (B'), sigma (S) and sometimes omega (W). Only the first three subunits are required for polymerase activity and are considered to be the core enzyme. There are also some additional B' units, the B' rudder and the B' lid which are involved in the security of the DNA strand in the holoenzyme. The remaining subunit, sigma factor, is required for RNA polymerase to bind to the promoter. With out sigma, or also the specificity factor, the enzyme only has a loose affinity for DNA, therefore only when the sigma factor is present will the initiation factor complex bind at the appropriate promoter location and sequence such as the -10 (TATAAT) and -35 (TTGACA) regions and move into the elongation process.  

Once the subunits have assembled the sigma factor is the first to interact with the promoter sequence. Interaction of the S subunit with a promoter signals the polymerase to initiate transcription at a specific sequence in template DNA. The β B and B′subunits polymerize ribonucleoside triphosphates (NTPs) as directed by the template strand. The A subunits interact with regulatory proteins and, in some cases with DNA to control how frequently RNA polymerase initiates transcription from a specific promoter. In order for this process to commence understanding of the atomic interaction of these units with the DNA during the first stages of initiation is crucial.

As the complex RF_c approaches the DNA attractive and repulsive binding interactions between the subunits and the DNA occur. Sigma is the first to interact and it forms a banana shape around the -10 box and loops through the top of the remaining subunits over to -35 where it forms more interaction. This is why under certain cellular stress conditions different sigma factors (1-6) are present, essentially allowing the interaction of different promoters on different genes coding for different proteins. Within the sigma subunit there are four regions of interest termed; 1.0 - 4.0.

Within the conserved region 2.2 - 3.0, what I called the banana, (Model 2) contains four distinct -10 promoter binding determinants. The first interacting group is in region 3.0. Exposed to the major groove of the DNA, His278 and Glu281 residues are linked to recognition of the -10 transcription element. His278 appears to play a more non-specific binding role being that it is too far away from the -10 but may link to -17 or -18, and Glu281 is within binding reach of T on the non-template strand. The second region of interest is located in sigma 2.4. This specialized region contains allele specific repressors of promoter mutations within the -10 element. Gln260 and (Asn or Thr)263 are also exposed to the major groove and thought to interact with -12.  Gln260 is easily within interaction reach of the -12 bases and could also interact with non-template strand T or template strand A. Small changes in the structure would allow (Asn or Thr)263 to interact with non-template strand T. The third DNA interaction group of sigma is in region 2.3. The highly conserved aromatic residues Phe248, Tyr253 and Trp256 play a role in promoter melting on the upstream edge forming the transcription bubble. The final -10 promoter interaction groups are located in regions 2.2 and 2.3. (2) There are two conserved basic residues. Arg237 and Lys241, both of which being positively charged residues are positioned to interact with the negative charged DNA in non-template positions -13, -14 and -15. Arg237 interacts with -13 or -14 and Lys241 interacts with -15.

The remaining element of sigma, region 4.0 which interacts with the -35 promoter. In order for the same subunit to interact with DNA that is nearly 20+ DNA residues away, it loops through the top part of the additional RNAP holoenzyme subunits and forms a helix-turn-helix fold region. Compelled around this recognition fold the DNA makes a 36° bend. Seven residues in the alpha subunit, which is broken up into its N-terminal (NTD) and C-terminal (CTD) ends also have interactions with DNA. A-CTD's that are most critical for DNA interaction and for UP element promoter function: L262, R265, N268, C269, G296, K298, S299 are just such residues.

So far these detailed interactions of sigma have been associated with the RF_o complex. Reviewing that the upstream region of DNA bends 36° around -35 due to the sigma 4.0 region of RNAP. At -16, DNA makes another bend, 37° turning toward the RNAP and the two strands then separate and take different paths beginning at -11 extending 15 residues downstream forming the transcription bubble. The newly formed single stranded non-template -10 element (-11through -7) region then crosses over sigma region 2.0. Where it can react with the exposed aromatic residues of 2.3. The strand from -2 through +4 is held by a groove between the two lobes of b1 and b2. The strand must now enter a tunnel enclosed on all sides by the protein. This tunnel is formed by parts of the sigma regions 2 and 3 and b1, the b¢ lid and the b¢ rudder. The interacting basic amino acids of sigma region 2.4 and 3.0 are exposed at the entrance of the tunnel essentially electro-statically pulling the template strand into the tunnel and moving it past the active site, which contains a magnesium ion. From +5 to +12 the strand is clamped between the b and b' subunits. The protein DNA interactions that cause the bending of the strand and the formation of the transcription bubble are what drive the transition from the closed inactive complex into the open active complex, containing the above conserved residues. The promoter binding regions from both polymerases contain important sequences and location (Figure 2) that are required for RNA polymerase to bind. These sequences are similar in both prokaryotic and eukaryotic genes, but the locations are different.

 Now that we have seen the Bpol RNAP in initiation action, a look at the eukaryotic RNA polymerase , pol II, complex can be examined. This eukaryotic pol II mRNA transcription protein complex (Model 2) is much more complex than the previous Bpol RNAP. Two large subunits and about 10 small subunits (Rpb’s) make up the core enzyme. The largest pol II subunit Rpb1, is homologous to beta' and the second largest subunit Rpb2, homologous to the beta subunit of Bpol.  Included in the core enzyme of pol II there are many non-polymerase factors that are required for binding of the enzyme to DNA.  These subunits find the promoter location and sequence at the -25 (TATA) and the -80 (CAAT) regions, which have been named TATA box and CAAT box respectively. Having the same end goal as the Bpol RNAP, that of synthesizing a transcript from a DNA template, we can combine our knowledge of the previous process to the one focused around pol II.

Examination of the parts and holoenzyme function of pol II (RNAPII) with comparison to Bpol we can gain understanding of how this remarkable system works. Like the sigma factor in Bpol there are a host of Rpb proteins that take the job in RNAPII.  To initiate the complex transcription factor IIF (tfg2 –yeast and Rap30 –humans) associates tightly with RNAPII forming the RNAPII-TFII complex. This complex shares clear sequence homology with Bpol’s sigma factor. (3) The addition of the TBP (tata box binding protein) and TFIIB, which have bound to the DNA mediator complex, institute what is known to be the catalytic core of RNAPII. TBP has binding association factors, TFIIA (like omega in Bpol) binds to TBP and the DNA, stabilizing the first interaction.

TFIIB binds between TBP and the location of the future Pol II binding site. Unlike Bpol, RNAPII has two essential initiation subunits. Edwards et al. in 1991 proved that without Rpb4 and 7 the initiation complex cannot bind to the DNA. Two additional general transcription factors, IIE and IIH, are required for the unwinding of the linear DNA but are not considered part of the core enzyme. The mediator complex contains hetero-dimer homologs from Bpol like the alpha and alpha prime, in RNAPII they are Rpb3 and Rpb11. Suggestions from Asturias (3) state that the mediator complex influenced by an activator will move from the closed complex, like in Bpol, into an open conformation allowing TFIIB and TBP to enter and conform into the complex. Once the TBP –TFIIB has entered the conformation changes once again allowing the RNAPII/IIF complex to bind initiating the closed promoter complex. The arrival of IIE and IIH factors shift the closed promoter complex into the open promoter complex, forming what is known as the scaffold complex.

Eukaryotes and prokaryotes are deemed to follow an abortive transcription and promoter escape phase before the elongation process begins. Abortive transcription initiation refers to the action of IIE and IIH factors in pol II, and sigma in Bpol, activating the promoter complex, RNAPII-IIF, to be opened and shift the coding DNA into the active site and stop at i and i+1 because of the binding interactions of the complex to the promoter sequences. All RNAPs reiteratively synthesize and release short RNA transcripts called abortives, (~2 to 9 nucleotides in length). Based on the structure of elongating eukaroytic RNAP II, Kornberg proposed that shorter nascent RNAs dissociate because they make fewer contacts with polymerase than do longer RNAs (4). Another explanation for these transcripts has emerged from the Murakami et al. structure (2002b) (1). Region 3.2 (sigma) occupies the RNA exit channel, leading to the speculation that these short RNAs must successfully compete with region 3.2 to continue to be retained in elongating polymerase complex. When RNA transcripts lose the competition, they are ejected as abortive transcripts; when they win in competition, region 3.2 is ejected and the transcript is successfully elongated. After the abortive transcription phase RNAPII-IIF escapes the promoter leaving behind a platform, (Scaffold complex) that rapidly facilitates assembly of a new pre-initiation complex and transcription re-initiation by a new RNAPII-IIF complex. These steps can only produce very short transcript products. In both initiation complexes as we have seen, move from a closed in-active conformation into an open active one through the process of the abortive and promoter escape process. Once these two steps have been completed the polymerase rejoins the complex and the process of elongation can begin. 

ELONGATION

Covalent addition of ribonucleic bases to the 3` end of a growing chain pairing with a single stranded DNA template.

 

Initial elongation also adopts abortive elongation intuitions much like in initiation. Marshall et al. in 1992 (Marshall et al. 1992) proposed a needed change in the holo-enzyme complex in order to move into full-blown elongation. They proposed the need for a positive transcription elongation factor or P-TEF. In 1995, (5) they found it and named it P-TEFb, also finding that P-TEFa and factor2 are stimulatory affecters in the synthesis of long transcripts. Additional information into the forwarding of transcription elongation came in 1996. Dahmas et al. proposed that phosphorylation of the CTD end of RNAPII was necessary for extended elongation. However, the mechanism for elongation in each RNAP is the same. The abortive initiation transcripts and the longer abortive elongation segments  after P-TEFb and TFIIH interact with the CTD end of RNAP the complex is in the right conformation to synthesize RNA from the DNA template in the active site containing an essential metal ion which is controlled by the interactions of the Rpb1 and 2 subunits. Activates nucleotides enter the complex and are orientated by the metal ion in order to coincide with the correct template base on the DNA. The DNA/RNA segment is split apart and goes its separate ways, RNA out past the rudder and lid loops and DNA back to re-anneal with the non-template strand but first orientate around the bridge helix and wall parts of the enzyme. The elongation syntheses of the transcript continues until a second pause region, or termination sequence is reached near the end of the template strand, shifting the structure into the termination mode.       

 

TERMINATION Recognition of the transcription termination sequence and the release of RNA polymerase and the newly formed RNA polymer.

 

In bacteria the termination sequence is followed by a model consistent with a protein mediated, rho, independent and dependant model for termination. In rho dependant termination rho binds to the formed RNA from the RNAP complex. It hydrolyzes ATP and moves 5' to 3' down the RNA toward the RNAP, rho eventually catches up with RNAP and melts the RNA-DNA duplex in the replication bubble. RNA is coiled inside of rho which pulls on the RNA that is inside RNAP causing termination by the dissociation of the RNA from RNAP. Rho-independent termination is caused by a  sequence of RNA that contains a G and C rich followed by a repeated U, A’s on the DNA template, to form a hairpin structure by the binding of the G and C nucleotides. The repeated U are at that time still inside the RNAP and are pulled on by the formation of this new structure causing the elongation complex to pause and eventually dissociate from the RNA. Therefore formation of this RNA secondary structure, called a hairpin, plays two roles in termination. If formed during rho-dependent termination it prevents rho from binding the RNA. And as we have just read, in rho-independent, it causes RNA polymerase to pause during transcription. Either case if this loop structure is formed or rho successfully winds the RNA termination can be accomplished.

 

MODEL 1

Bacterial RNA polymerase from Thermus aquaticus (pdb code 116V)

Bacterial RNAP conitains many homologous sunbuntis in comparision to eukaryotic RNAP. Seen in MODEL 2