• RNA polymerase II
• References
• Images
• Slides

           RNA polymerase II (Pol II) from Saccharomyces cerevisiae (PDB ID: 1Y77) is a protein of critical significance as essentially all living organisms have a variation of this protein.  RNA polymerase II facilitates the transcription of DNA into RNA which leads to creation of all proteins within an organism. Creation of proteins is an important biological function as without it the organism cannot function and life would not exist. Pol II has a molecular weight of 516,293 daltons and an isoelectric point is 5.51 (1). Pol II has 12 subunits and the associated ligands of this molecule include phosphomethylphosphonic acid guanylate ester (G2P), magnesium ion and zinc ion (2).

            RNA polymerase II which consists of 12 subunits is responsible of transcribing DNA into messenger RNA (mRNA) in three stages of initiation, elongation and termination (3). RNA polymerase II elongation complex is predominant in the elongation stage during which the subunits of Pol II, which include secondary structures such as beta sheets, alpha helices, 3/10 helices and random coils, are able to unwind the DNA strand into its sense and nonsense strand, bind to the template DNA strand and synthesize a complementary mRNA strand. A specific hydrogen bonding interaction occurs in the active site of Pol II between an acidic Asn-479 residue (in subunit RpB1), and 2’-OH group of the ribose moiety of nucleoside triphosphate (NTP) which enables the protein to discriminate between NTP and desoxy-NTP (2).  Pol II is connected to RNA/DNA complex and has 3 different types of ligands which are phosphomethylphosphonic acid guanylate ester (G2P), zinc and magnesium ions. G2P is a prosthetic group which is similar in to the nucleoside triphosphate, guanosine-5'-triphosphate (GTP). GMPCPP is a non-reactive analogue of G2P that allows the crystallographers to accurately determine the ligand binding site of G2P. The residues that inteact in hydrogen bonding with G2P binding pocket are Asp-481, Asp-483, Asp-485, Asn-479, Arg-1020, and Arg-766. As depicted in the binding pocket slide, the magnesium ion binds to the nucleoside triphosphate moiety (3). The NTP substrates are linked by Pol II in specific sequence based on the DNA strand to form the mRNA (4). Pol II moves along the DNA strand in 3’ to 5’ direction so RNA is transcribed in the 5’ to 3’ direction (4). The Pol II elongation complex is attached to a DNA/RNA hybrid and has magnesium ions and zinc ions as its associated metal ions. The role of the metallic ligands is suggested to be in use during catalytic nucleotide incorporation. 

Several functionally important residues allow Pol II to function. A majority of these residues are present in the active site of Pol II which is composed of subunits Rpb1 and Rpb2 (5). A collection of glutamic acid residues (Glu-1403, Glu-1404, and Glu-1407) collectively repel the DNA strand in conjunction with basic residues (Arg-326, Lys-330, Arg-337) that pull the template strand. These residues collectively cause enzyme-induced distortion and destabilization of the incoming DNA duplex that may drive strand unwinding into an upstream and downstream strand (2). The acidic Asp-290 residue helps the TFISS, an elongation factor, hairpin to activate a nucleophilic water molecule which is hypothesized to help TFIIS induce a shift of the RNA strand (this residue is mutated to an acidic glutamate residue in the molecular slide). The TFISS hairpin is located near the bridge helix. Realignment of the RNA in the active site allows for optimized RNA cleavage. The general role of TFIIS in Pol II is to restart an arrested Pol II during transcription. The residue Glu-291 assists the TFIIS hairpin to perform RNA cleavage and to bind NTPs. In addition to these important residues a series of residues create prominent loops called the fork loop 1, fork loop 2, rudder and lid.

The lid is involved with the maintenance of the upstream end of the DNA/RNA hybrid and plays a steric role in DNA-RNA strand separation as its residues do not contact separating nucleic acids. The lid helps from a RNA exit tunnel that allows only a single strand to pass thus enabling separation.  The rudder and fork loops have a prominent role during the initiation-elongation transition of Pol II as they create two compartments that are able to hold the DNA/RNA hybrid and the downstream DNA.  These strands have a very important function in the active site of Pol II but the composition of the residues that make up these is poorly conserved and vary in length, thus suggesting that the role of these loops is purely due to steric interactions (2).  These prominent loops in the Pol II structure give rise to an overall Pol II bubble-RNA complex, which is the tertiary structure of the enzyme when it is synthesizing mRNA. The bubble complex as illustrated in Figure 1, gives rise to a cleft in the enzyme molecule. The entering DNA forming a bridge helix moves to the active site at the core of molecule. The synthesis of mRNA molecule occurs near the hybrid compartment and the RNA molecule exits the polymerase at ether of the exit sites labeled 1 and 2 near the dock element of the enzyme

            RNA polymerase II with human VP16-Mediator-poll II-TFIFF assembly (PBD ID: 3J0K) from Homo sapiens is a comparable protein to Pol II from Saccharomyces cerevisiae, Baker’s Yeast, as it is essentially the same protein functioning in a different species. This comparison was chosen protein based on the results of two servers, PSI-Blast and Dali Server, which function to find protein structures that are similar to the protein of interest. Position-Specific Iterated Basic Local Alignment Search Tool (PSI-Blast) compares the amino acid sequence of the protein of interest against a wide array of protein databases (6). But the Dali server uses a program called DaliLite v.3 to compare tertiary structures of proteins that may be structurally similar but are not detected via comparison of primary structure (7). PSI-Blast compared Pol II from Saccharomyces cerevisiae (protein of interest) to produce an E-value which is a measure of similarity between the protein of interest and a similar structure. An E-value below 0.05 is statistically significant to conclude that the two proteins are similar in primary structure. The Dali server search produces Z-scores which if are above the threshold of 2.0 are significantly similar (7). RNA polymerase II from Homo sapiens had a PSI Blast E value of 0.0 and a Z score of 40.5 (8, 9). 

            The PSI-Blast and Dali servers’ results for the RNA polymerase II from Homo sapiens indicate that both polymerase are essentially the same molecule, but in different species. Every comparison protein suggested by the results of the PSI-Blast and Dali servers’ was a derivation of the same polymerase II molecule found with a different ligand or sometimes in a different species. The lack of variance in results can be attributed to the fact that the highly specialized and important role of polymerase II leads to a highly conserved and near ubiquitous presence across all living organisms. The biggest difference between the protein of interest and comparison protein is the molecular weight as comparison protein’s molecular weight is only 468,783 daltons despite also being a 12 subunit protein and having the same number of residues (1). The comparison protein also lacks a nucleoside triphosphate ligand such as G2P found in the protein of interest. The comparison protein’s structure also consists of a human mediator complex and a variety of factors such as TFFIA, TFFIB and TFIIF, collectively referred to as Pre-Initiation   Complex (PIC), which are not present in the protein of interest (10). This difference suggests that method RNA polymerase II across different species requires unique coenzymes or ligands to function despite having almost the same primary, secondary and tertiary structure. Another significant difference between the two molecules is the specificity of the role that they play as the protein of interest is elongation complex of polymerase II while the comparison protein is specifically crystalized to study initiation complex. This leads to hypothesis that tertiary structure of the polymerase does not vary to a great extent between the initiation complex and the elongation complex.