RNA polymerase II

RNA polymerase II (RNAP II and Pol II) is a multiprotein complex. It is one of the three RNAP enzymes found in the nucleus of eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to upstream gene promoters and begin transcription. RNA polymerase II (RNAP II and Pol II) is a multiprotein complex. It is one of the three RNAP enzymes found in the nucleus of eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to upstream gene promoters and begin transcription. Early studies suggested a minimum of two RNAPs: one which synthesized rRNA in the nucleolus, and one which synthesized other RNA in the nucleoplasm, part of the nucleus but outside the nucleolus. In 1969, science experimentalists Robert Roeder and William Rutter definitively discovered an additional RNAP that was responsible for transcription of some kind of RNA in the nucleoplasm. The finding was obtained by the use of DEAE-Sephadex ion-exchange chromatography. The technique separated the enzymes by the order of the corresponding elutions, Ι,ΙΙ,ΙΙΙ, by increasing the concentration of ammonium sulfate. The enzymes were named according to the order of the elutions, RNAP I, RNAP II, RNAP IΙI. This discovery demonstrated that there was an additional enzyme present in the nucleoplasm, which allowed for the differentiation between RNAP II and RNAP III. The eukaryotic core RNA polymerase II was first purified using transcription assays. The purified enzyme has typically 10–12 subunits (12 in humans and yeast) and is incapable of specific promoter recognition. Many subunit-subunit interactions are known. RPB3 is involved in RNA polymerase II assembly. A subcomplex of RPB2 and RPB3 appears soon after subunit synthesis. This complex subsequently interacts with RPB1. RPB3, RPB5, and RPB7 interact with themselves to form homodimers, and RPB3 and RPB5 together are able to contact all of the other RPB subunits, except RPB9. Only RPB1 strongly binds to RPB5. The RPB1 subunit also contacts RPB7, RPB10, and more weakly but most efficiently with RPB8. Once RPB1 enters the complex, other subunits such as RPB5 and RPB7 can enter, where RPB5 binds to RPB6 and RPB8 and RPB3 brings in RPB10, RPB 11, and RPB12. RPB4 and RPB9 may enter once most of the complex is assembled. RPB4 forms a complex with RPB7. Enzymes can catalyze up to several million reactions per second. Enzyme rates depend on solution conditions and substrate concentration. Like other enzymes POLR2 has a saturation curve and a maximum velocity (Vmax). It has a Km (substrate concentration required for one-half Vmax) and a kcat (the number of substrate molecules handled by one active site per second). The specificity constant is given by kcat/Km. The theoretical maximum for the specificity constant is the diffusion limit of about 108 to 109 (M−1 s−1), where every collision of the enzyme with its substrate results in catalysis. In yeast, mutation in the Trigger-Loop domain of the largest subunit can change the kinetics of the enzyme. Bacterial RNA polymerase, a relative of RNA Polymerase II, switches between inactivated and activated states by translocating back and forth along the DNA. Concentrations of eq = 10 μM GTP, 10 μM UTP, 5 μM ATP and 2.5 μM CTP, produce a mean elongation rate, turnover number, of ~1 bp (NTP)−1 for bacterial RNAP, a relative of RNA polymerase II. RNA polymerase II is inhibited by α-Amanitin and other amatoxins. α-Amanitin is a highly poisonous substance found in many mushrooms. The mushroom poison has different effects on the each of the RNA Polyermases: I, II, III. RNAP I is completely unresponsive to the substance and will function normally while RNAP III has a moderate sensitivity. RNAP II, however, is completely inhibited by the toxin. Alpha-Amanitin inhibits RNAP II by strong interactions in the enzyme's 'funnel', 'cleft', and the key 'bridge α-helix' regions of the RPB-1 subunit. RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. It consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. Part of the assembly of the holoenzyme is referred to as the preinitiation complex, because its assembly takes place on the gene promoter before the initiation of transcription. The mediator complex acts as a bridge between RNA polymerase II and the transcription factors.