Thermus aquaticus RNA Polymerase
Created by Robert Schenck
Thermus aquaticus is a thermophilic bacterium. This species' protein RNA polymerase is a prototypical DNA-dependent RNA polymerase (RNAP) with a theoretical iso-electric point and molecular weight of 5.80 and 547754.35 g/mol, respectively. Thermus aquaticus RNAP (Taq RNAP) is a key enzyme involved in gene expression at the stage of transcription. Taq RNAP is classified as a transferase, responsible for polymerizing ribonucleotides (Image 1). Furthermore, Taq RNAP is a major target for gene regulation (6). When associated with a sigma factor, Taq RNAP can bind to a 40 to 60 base pair region of duplex promoter DNA. A sigma factor is a prokaryotic transcription factor that enables specific binding of RNAP to gene promoters. The RNAP-sigma complex rips open the double helix and places the template DNA into the Taq RNAP active site. Once the DNA is opened, the Taq RNAP-DNA complex makes a conformational change to a more stable transcription elongation complex. Following, extensive RNA synthesis can proceed at the active site. Upon completion, the RNA-DNA hybrid would separate near a feature called the "rudder." Collectively, RNA polymerases synthesize nearly all cellular RNA molecules.(4) Without Taq RNAP, Thermus aquaticus would be unable to transcribe its DNA, leaving it unable to translate proteins. The organism would be unable to function.
Thermus aquaticus RNA polymerase exists as a dodecamer with
four major subunits: alpha, beta, beta prime, and omega. The protein takes the overall shape of a
"crab claw." The beta subunit makes the upper portion of the jaw. The lower jaw is composed of the beta prime subunit. Collectively, they compose the DNA channel. The DNA channel is 27 Å wide. The residence of an Mg2+ ion at the back wall of this internal channel, where there is much interaction between the beta and beta prime subunits, is necessary for catalytic activity. Furthermore, a secondary channel forms that is only 10-12 Å wide, not large enough for double stranded DNA or DNA-RNA to fit. The alpha and omega subunits appear elsewhere in the protein and have different functions. The alpha subunit is responsible for binding catabolite activator protein (CAP), which is a protein important for transcription activation. The omega subunit may be a chaperonin and aid RNA polymerase (RNAP) assembly.(7)
Random coils, beta sheets, turns, and alpha helices appear in each subunit. However, the alpha helix is both the most prevalent and functionally important secondary structure in the protein. For example, the beta prime subunit has 41 helices and 63 helix-helix interactions, but only 20 beta sheets.(7) As discussed later in more detail, these helices form a
coiled-coil that proves to be functionally important.
In order for Taq RNAP to polymerize ribonucleotides from DNA, the enzyme must bind both DNA and the nucleotide triphosphates that will add to the synthesizing RNA chain. Many of these binding sites and other functionally important residues appear inside of the internal channel near the Mg2+ active center site. The
binding pocket for the Mg2+ occurs at residues
Asn-737, Ala-738, Asp-739, Phe-740, Asp-741, Gly-742, and Asp-743 in the beta prime chain. These residues are absolutely conserved among bacterial RNAP. Collectively, the aspartic acid residues provide this pocket with a
formal charge of -3 making it a good site for the positively charge magnesium ion.(8)
Taq RNAP can bind two nucleoside triphosphates (NTPs) at a time. They bind at sites known as the i site and the i + 1 site. The i site becomes the 5' end of the RNA transcript. The i + 1 site, sometimes referred to as the elongation site, extends the i site nucleotide in the 3' direction upon formation of the phosphodiester bond. Three important basic residues for binding the negatively charged NTP, Lys-838, His-999, and Lys-1004, appear on the beta subunit clustered on the back wall of the internal channel. This binding all occurs within 11 Å of the active site.(8)
The Taq RNAP does not directly bind to DNA. Rather, it requires a holoenzyme complex with a sigma cofactor. The three domains of the sigma cofactor lie across the upstream face of the RNAP, wind through the active site and out of the RNA exit channel. The sigma shares a large interface with the coiled coil region of the beta prime subunit. The sigma factor contains many important functional residues for the overall protein complex. His-278 and Glu-281 of the alpha helix bind to the DNA promoter. Glu-281 may interact with the nontemplate strand T at-13 while His-278 appears to interact with the phosphate backbone of the nontemplate strand at positions -17/-18. Aromatic residues Phe-248, Tyr-253 and Trp-256 play a role by interacting with the promoter at least partly through sequence-specific binding of the nontemplate strand of the melted -10 element. The -10 element refers to the mostly single stranded DNA at the fork junction. These residues seem ideally positioned to interact with unpaired bases of the single-stranded tail of the nontemplate strand DNA, which crosses the surface-exposed aromatic residues. The numbered system used above refers to the DNA position with respect to the transcription start site at +1. Finally, the positively charged residues Arg-237 and Lys-241 interact with the negatively charged DNA backbone and lead it into the main channel.(5)
It is hypothesized that when the sigma factor binds, the RNAP undergoes a conformational change that can open and close the main channel by distances greater than 20 Å. Although not completely solved, the most accepted theory states that a series of isomerization steps yields a transcription-competent open promoter complex. During the isomerization process, fourteen DNA base pairs are melted. If nucleotide substrates are also present, then RNA synthesis can begin.(6)
Other conformational changes may occur in the presence of certain drugs. Sorangicin and Rifampicins are known to inhibit the elongation process after polymerization of two to three nucleotides. Both drugs use the same binding pocket on the beta chain: Val-137, Gln-390, Gln-393, Phe-394, Asp-396, Arg-405, His-406, Arg-409, Ser-411, Pro-444, and Ile-452.(2) Originally it was thought that these drugs sterically clashed with RNA chains longer than two or three nucleotides in length. However, new theories believe that this may not be enough to explain the dramatic buildup of two and three length nucleotide chains. These drugs may send an allosteric signal to the RNAP that makes binding to Mg2+ unfavorable. Dissociation from the catalytic magnesium slows down the reaction and results in many aborted short fragments.(1) Sorangicin and Rifampicin are known to effectively block other bacterial RNAPs. Thus, it is no surprise that these drugs are effective on similarly structured proteins such as the Thermus thermophilus RNAP. RNA polymerase from Thermus thermophilus (PDB ID: 2A68) received a score of 16.8 when compared to Taq RNAP on the Dali Server. This database output exhibits the significant structural similarity among these proteins.(3) Additionally, when both the beta and beta prime subunits of Thermus Thermophilus RNAP were primary sequence aligned using the program BLAST, the program returns an E-value of 0.0, making them equivalent. The alpha chains produce an E-value of 5e-145, and the omega chains have an E-value of only 3e-52. According to BLAST data, the chains decrease in similarity as they beocome less functionally important. However, these scores indicate that the overall sequence of these proteins are extremely similar. It may be important to note that Thermus thermophilus RNAP has a fifth unique chain that Taq RNAP does not contan. Nevertheless, the Thermus thermophilus RNAP performs an identical function as Taq RNAP: polymerizing ribonucleotides for the syntheis of RNA molecules from DNA. Thus, unsurprisingly there is strong evidence that these two proteins have very similar binding pockets. (1)
Taq RNAP also binds to the metal ion Zn2+. The zinc binding motif consists of four cysteine residues: Cys-1113, Cys-1195, Cys-1201, Cys-1204. These residues are absolutely conserved in bacterial RNAPs. Based on its location on the bottom side of the beta prime subunit outside of the channel, the Zn2+ is hypothesized to aid in folding.(8)
Collectively, the crab claw structure of Taq RNAP provides a complex system that can efficiently synthesize RNA chains.