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

• Published in: The Journal of biological chemistry Vol. 274; no. 8; pp. 5104 - 5113
• Main Authors: Wlassoff, W A, Kimura, M, Ishihama, A
• Format: Journal Article
• Language: English
• Published: United States 19.02.1999
• Subjects: Base Sequence; Catalytic Domain; DNA; Hydrolysis; Photoaffinity Labels; Photochemistry; RNA Polymerase II - chemistry; RNA Polymerase II - metabolism; Schizosaccharomyces - enzymology; Schizosaccharomyces pombe; Transcription, Genetic; Uridine Triphosphate - analogs & derivatives; Uridine Triphosphate - biosynthesis
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.8.5104

The catalytically competent transcription complex of RNA polymerase II from the fission yeast Schizosaccharomyces pombe was affinity labeled with photoreactive nucleotide analogues incorporated at 3' termini of nascent RNA chains. To locate the catalytic site for RNA polymerization, the labeled subunits 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 subunits, Rpb1 and Rpb2, with the most available sites of proteolysis in junctions between the conserved sequences among RNA polymerase from both prokaryotes 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 regions 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 catalytic site. All these regions include the conserved sequences of RNA polymerases, and the catalytic site of Rpb2 belongs to an NH2-terminal part of its conserved sequence H.