Functional Organization of Two Large Subunits of the Fission Yeast Schizosaccharomyces pombe RNA Polymerase II

Abstract

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 NH 2 -terminal part of its conserved sequence H.Eukaryotic RNA polymerase II is a multifunctional and multisubunit enzyme consisting of more than 10 putative subunits (1, 2). In contrast to prokaryotic RNA polymerases, little is known about the molecular architectures of eukaryotic counterparts except the following: (i) the two largest subunits, homologous to bacterial ␤ and ␤Ј subunits, are involved in the binding of DNA template, the polymerization of RNA chains, and the association of nascent RNA chains (1, 2); and (ii) the subunit 3, a homologue of bacterial ␣ subunit, plays a role in the assembly of RNA polymerase (3). Knowledge of the structure and function of the individual subunits is essential for understanding of the molecular mechanisms of transcription and regulation of the protein-coding genes in eukaryotes. Toward this ultimate goal, we have studied the structure-function relationship of Schizosaccharomyces pombe RNA polymerase II.The S. pombe RNA polymerase II is composed of 11 subunits lacking subunit 4 (4, 5). Analyses of the subunit-subunit contact network using different approaches indicate that the two large subunits provide the platform for assembly of other small subunits (6 -9). In this study, we tried to locate the active center for RNA polymerization using the method of affinity labeling with photoreactive nucleotide analogues. This technique has been used to identify the active sites of Escherichia coli RNA polymerase (10 -13), calf thymus RNA polymerase II (14), Saccharomyces cerevisiae RNA polymerase I(A), II(B), and III(C) (15-18), HeLa RNA polymerase II (19), wheat germ RNA polymerase II (18, 20), and influenza virus RNA polymerase (21). Depending on experimental systems such as the type of enzymes and templates, the structure of affinity reagents and the length of nascent RNA chains, the cross-linking has been observed at differen...

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

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Thrombospondin and fibrinogen bind serotonin-derivatized proteins on COAT-platelets

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Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair

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