Enzyme

There are three subforms of RNA Polymerase II. Initially, Kedinger and colleagues named the forms B0, BI, BII. Currently they retain the names IIo, IIa, and IIb, a nomenclature used by Schwartz and Roeder. Kedinger and colleagues resolved RNA polymerase II into BI and BII (IIa and IIb) from rat liver and calf thymus using denaturing polyacrylamide gel electrophoresis. Schwartz and Roeder were able to resolve all three forms of the enzyme from mice also using denaturing gel electrophoresis.

Analysis of gel migration distance revealed that IIb moved the farthest, followed by IIa and IIo. Thus RNA polymerase IIb had the lowest molecular weight and IIo, the highest. Apparently variation within a particular subunit determined the gel mobility of the enzyme. This defining subunit was RPB1, which contained a carboxyl-terminal domain (CTD). IIb appeared to missing this critical part of RPB1, which was attributed to proteolytic destruction during purification in vitro (Sentenac 1990;Weaver 1999). Its absence in IIb was confirmed by Laybourn and Dahmus because monoclonal antibodies directed against the subunit in subform IIo and IIa did not attach to IIb. The antibodies attached to the conserved heptapeptide repeat found on the carboxyl-terminal domain of RPB1:

Tyr-Ser-Pro-Thr-Ser-Pro-Ser They also found that the CTD was involved in initiation and elongation. The antibodies also bound to the unphosphorylated form (IIa) of the enzyme with more affinity and inhibited initiation of transcription. The antibodies were bound to the phosphorylated form (IIo), which was involved in elongation.

The enzyme is the most studied of the polymerases present in the cell because it is primarily responsible for the transcription of mRNA of cytosolic proteins. It is also responsible for transcription of snRNAs, (except the U6 gene) and heterogenous nuclear RNAs (which may sometimes be precursors of mRNAs) (Weaver 1999). S. cerevisiae RNA polymerase II has been purified by immunoprecipitation using monoclonal antibodies against specific subunits and each subunit has been resolved using SDS-PAGE (Young 1991, 1998; Weaver 1999). The enzyme preferentially initiates transcription at promoter regions in highly supercoiled templates with an average rate of chain elongation at 60 to 600 nucleotides per minute in vitro (Sentenac 1990).

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