Dissociation of two polypeptide chains from yeast RNA polymerase A.

Abstract

Yeast RNA polymerase A (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) can be converted to a new form of enzyme, called RNA polymerase A*, which is lacking two polypeptide chains of 48,000 and 37,000 daltons. Apart from these two missing polypeptides the subunit structures of RNA polymerases A and A* are indistinguishable. RNA polymerase A* differs from the complete enzyme in its electrophoretic and chromatographic behavior, template requirements, and aamanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T). with the same efficiency as RNA polymerase A but its specific activity is greatly reduced with native calf thymus DNA as template. The transcription of a variety of synthetic templates is also altered by removal of the two polypeptide chains. RNA polymerase A* is inhibited by high concentrations of a-amanitin (500 Mg/ml), whereas RNA polymerase A is comparatively less sensitive to the toxic peptide. The data are discussed in terms of possible roles of the two dissociable polypeptides.There is currently considerable interest in the structural and functional properties of the multiple forms of RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) isolated from eukaryotic cells. Several laboratories have purified these enzymes from various organisms (1). Nuclear RNA polymerases. are quite complex multimeric proteins which appear to consist of two large subunits in equimolar amount associated with a series of smaller polypeptide chains (2-6). This structural complexity suggests the likelihood that each enzyme form is made of a fundamental enzyme surrounded with regulatory components or specificity determinants having a specialized role in transcription. Although no available evidence yet supports this hypothesis, past experience with other polymerizing enzymes shows that the basic templatedirected polymerization reaction can be carried out by relatively simple proteins (7-9). If small polypeptide chains present in the RNA polymerase molecule were indeed endowed with regulatory function one could expect some of them to be loosely and reversibly associated to the basic enzyme.As our experience with yeast RNA polymerase A accumulated, persistent peculiarities were noted during fractionation which indicated that the enzyme could be resolved into two distinct enzymatic fractions. It is the purpose of this report to detail the experiments which exploited these hints. The data show that two polypeptide chains of 48,000 and 37,000 daltons are reversibly associated to RNA polymerase A. Following gel electrophoresis or phosphocellulose chromatography RNA polymerase A can be converted to RNA polymerase A*, which is lacking these two satellite proteins. RNA-polymerase A* differs from the complete enzyme in its template requirements as well as other properties. A preliminary report of this work has been published (10). MATERIALS AND METHODSNucleic Acids and RNA Polymerases. Synthetic polymers w...

Functional architecture of RNA polymerase I

“…Heterodimerization of A49 and A34.5 explains why the two subunits dissociate together from Pol I upon urea treatment (Huet et al, 1975), why Pol I purified from a yeast strain lacking the gene for A34.5 also lacks A49 (Gadal et al, 1997), and why two distantly related mammalian Pol I subunits bind each other (Yamamoto et al, 2004). It is also consistent with the observed continuous EM (Armache et al, 2005) (gray) into the EM density (blue).…”

“…To confirm this assignment, we dissociated subunits A49 and A34.5 from Pol I with the use of urea (Huet et al, 1975), purified the resulting 12-subunit variant Pol I DA49/34.5 (previously called Pol A* [Huet et al, 1975]), and solved its structure by cryo-EM at 25 Å resolution (Figures 4A and 4B; Experimental Procedures). The structure was similar to the complete Pol I, except that the density assigned to A49 and A34.5 was lacking ( Figure 4B).…”

The role of basic proteins in the DNA dependent RNA polymerase reaction

“…This finding, together with the aforementioned affinity between the fraction S activity and the RNA polymerase B, raised the question of whether the fraction S activity may not simply represent an easily dissociable enzyme subunit which thus could in some cases become separated from the actual enzyme molecules. Further support for this hypothesis was provided by recent findings, that mammalian enzymes in fact may lose a part of their smaller subunits; this was demonstrated for the RNA polymerase A of calf thymus [S] as well as of yeast [6].…”

Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity

“…Therefore, although one might be inclined to think in more exclusive terms of RNA polymerase I-and/or III-like or RNA polymerase IT-like activities when considering viroid replication, the action of a minor yet distinct RNA-synthesizing system must still be entertained. Reports of atypical polymerase activities such as a-amanitinsensitive polymerase I activity (15), electrophoretic variants of polymerase II, and rRNA-encoding DNA transcription by polymerase 11 (16) Alternatively, the properties we have described here may simply reflect a process involving a total lack of polymerase specificity, with all nuclear polymerases competent in transcribing a truly unusual RNA structure. Since RNA polymerase II exists in the highest concentration in vivo and is potentially most available in the nucleoplasm, it may emerge as the polymerase of choice for CEV synthesis.…”