Functional organization of two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II - Location of the catalytic sites

The catalytically competent transcription complex of RNA polymerase II from the fission yeast Schizosaccharomyces pombe was affinity labeled with phot oreactive nucleotide analogues incorporated at 3' termini of nascent RNA ch ains. To locate the catalytic site for RNA polymerization, the labeled subu nits were separated by SDS-polyacrylamide gel electrophoresis and subjected to partial proteolysis, After microsequencing of proteolytic fragments, a complex multidomain organization was indicated for both of the two large su bunits, Rpb1 and Rpb2, with the most available sites of proteolysis in junc tions between the conserved sequences among RNA polymerase from both prokar yotes and eukaryotes. The cross-linking studies indicate the following: (i) the 3' termini of growing RNA chains are most extensively cross-linked to the second largest subunit Rpb2 between amino acids 825 and 994; (ii) the r egions 298-535 of Rpb2 and 614-917 of Rpb1 are cross-linked to less extents , suggesting that these regions are situated in the vicinity of the catalyt ic site. All these regions include the conserved sequences of RNA polymeras es, and the catalytic site of Rpb2 belongs to an NH2-terminal part of its c onserved sequence H.