Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

Abstract

Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000,82,000,41,000, 40,500, 37,000, 34,000,28,000,24,000,20,000,14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits.As In lower eukaryotes, however, the situation has remained unclear. Although the original studies of Roeder and of Rutter and coworkers (9) and later of others (10-12) reported the chromatographic evidence for the presence of polymerase III in yeast, there has never been a confirmation by isolation and characterization of the structure of this enzyme in any lower eukaryotes. In contrast, polymerases I and II have been isolated and their subunit structure defined for yeast (13, 14, t), Dictyostelium (15), and Physarum (16).In this paper we report the characterization of yeast polymerase III which can be isolated by two different procedures. The homogeneous preparation contains two large polypeptides (160,000 and 128,000 daltons), one polypeptide of intermediate size (82,000 daltons), and 9 to 10 smaller peptides with stoichiometries varying from 1 to 2 moles per mole of enzyme. This structure is clearly different from yeast polymerases I and II and bears a general resemblance to polymerases III from Xenopus and mouse plasmacytoma. These results provide a convincing argument for the existence of class III polymerases in all eukaryotes and establish a structural basis for the discrimination of the three enzymes.MATERIALS AND METHODS Materials. Materials and yeast cells were obtained as described previously (13). DNA-cellulose was prepared by the method of Alberts and Herrick (17), using denatured calf thymus DNA.RNA Polymerase Assay. Enzymes were assayed as described previously (13). One unit corresponds to the incorporation of 1 nmol of UMP into RNA in 10 min at 300.Enzyme Purification. Yeast RNA polymerase III was purified to homogeneity by two methods. Method A: Polymerase III was obtained as a by-product of a large-scale purification of yeast polymerase I. The first three steps are batchwise treatment with phosphocellulose, batchwise treatment with DEAE-cellulose, and ion-filtration chromatography on DEAE-Sephadex. The details of these procedures, which are common to the purification of polymerase I, will be published elsewhere (13). Enzymes I and III are then separated by DNA-cellulose chromatography (Fig. 1). Whereas, enzyme I is eluted at 0.45 M KCI, polymerase III binds more stron...

Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

“…The mouse and yeast subunits differ at the site of mutation in what otherwise would be a string of 11 consecutive identical residues. Yeast RNA polymerase II is 500 times less sensitive to cx-amanitin than the mouse enzyme (55), and it is possible that this single difference (asparagine in mouse, serine in yeast) is the cause of the greater resistance of the yeast enzyme.…”

Synthesis of Ribosomal and Transfer Ribonucleic Acids in Yeast During a Nutritional Shift-up

“…It is probable that in yeast, by analogy with higher eukaryotes (Weinmann & Roeder, 1974), the 25S, 18s and 5.8s rRNA species are synthesized by RNA polymerase I and the 5 S rRNA and 4 s tRNA are synthesized by different forms of RNA polymerase 111. The activities of these polymerases could be co-ordinated by them all requiring a common sub-unit (Valenzuela et al, 1976). The difference in accumulation rate may be due to the component having a lower affinity for the form of RNA polymerase 111 synthesizing tRNA.…”

Transcription Termination by RNA Polymerase III in Fission Yeast

“…5D) as previously reported for the S. pombe 7SL RNA gene (54) (note that the concentrations used here may not reveal an accurate curve for the human enzyme). Since S. pombe and human pols III are sensitive to ␣-amanitin and S. cerevisiae is not, inhibition by this toxin provides another criterion that distinguishes these enzymes (55). DISCUSSION We have developed a tRNA suppressor gene whose biological function in S. pombe is dependent on accurate and efficient termination by pol III, and we used it to study transcriptional termination in vivo and in vitro.…”