Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.     

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P_i in the presence of manganese or cobalt (K_m = 19 µM ATP; k_cat = 67 s^–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I_0.5 = 1 mM), pyrophosphate (I_0.5 = 0.4 mM) and tripolyphosphate (I_0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.     

Crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe RNA polymerase III

Eukaryotic RNA polymerase III (Pol III) is a multisubunit enzyme responsible for transcribing tRNA, 5S rRNA, and several small RNAs. Of the 17 subunits in Pol III, the C17 (Rpc17) and C25 (Rpc25) subunits form a stable subcomplex that protrudes from the core polymerase. In this study, we determined the crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe. The subcomplex adopts an elongated shape, and each subunit has two domains. The two subunits in the subcomplex are tightly packed and extensively interact, with a contact area of 2080 Ų. The overall conformation of S. pombe C17/25 is considerably different from the previously reported structure of C17/25 from Saccharomyces cerevisiae, with respect to the position of the C17 HRDC domain, a helix bundle essential for cell viability. In contrast, the S. pombe C17/25 structure is quite similar to those of the Pol II and archaeal counterparts, Rpb4/7 and RpoE/F, respectively, despite the low sequence similarity. A phylogenetic comparison of the C17 subunits among eukaryotes revealed that they can be classified into three groups, according to the length of the interdomain linker. S. pombe C17, as well as Rpb4 and RpoF, belongs to the largest group, with the short linker. On the other hand, S. cerevisiae C17 belongs to the smallest group, with the long linker, which probably enables the subcomplex to assume the alternative conformation.