No abstract

The molecular structure of calf thymus RNA polymerase AI, BI and B I I has been analyzed. The data are consistent with subunit models of [(SAl, N r 197000), (SA2, Mr 126000), (SA3, Mr 51000)1 (SA4, Mr 44000)i (SA5, Mr 25000)z (SA6, ill, 16500)2), [(SBl, Mr 214000)1 (SB3, M , 14OOOO)1 (SB4, Mr 34OOO)1-, (SB5, Mr 25OOO)z (SB6, Mr 16500)3-,j, [(SB2, Nr 180000), (SB3, Nr 140000), (SB4, Mr 35000),-, (SB5 Mi-25000), (SB6, M , 16500)3-4] for enzyme AI, B I and B 11, respectively. Enzyme B I I was further resolved by polyacrylamide gel electrophoresis into two forms, BIIa and BIIb, which seem to differ only in the charge of their largest subunit, SR2a and SBSb, respectively. Polyacrylamide gel in the presence of urea has shown that SB6 subunit could be split into two components, SB6a and SB6b, of similar molecular weight, but of different charge. It appears that A1 and B enzymes contain two couples of small subunits of identical molecular weight and charge, SA5 and SB5, and SA6 and SB6a.Sedimentation through glycerol gradients suggest values of 14.5 S and 15.5 S for the sedimentation constants of calf thymus enzymes A1 und B, respectively. These values are consistent with the estimation of their molecular weight by electrophoresis in acrylamide gels of graded porosity: 550000 -fr loo/,, 600000 & loo/, and 570000 5 loo/, for enzymes AI, BI and BII, respectively.Rat liver RNA polymerase B activity most probably corresponds to a mixture of three different enzymes. Two of them, BI and BIIb, have subunit composition identical to their calf thymus counterparts, while the third one (BO) differs from the other B enzymes by the size of its largest subunit only. It does not seem that the multiple calf thymus and rat liver B enzymes correspond to artifacts in vitro, arising during purification by proteolysis of the largest subunit of a unique RNA polymerase B.Antibodies to calf thymus enzyme A1 were prepared. The antiserum specifically precipitated calf thymus enzyme AI and failed to give precipitation lines with either calf thymus B or Escherichia coli RNA polymerases. I n addition, the crude antiserum of the partially purified y-globulin fraction completely inhibited partially purified or pure calf thymus enzyme A1 preparations, while pure calf thymus enzymes B or E . coli RNA polymerase were not inhibited. However, partially purified calf thymus enzymes B were significantly inhibited. Cross-reactions were also observed with partially purified enzymes A1 and B from rat liver and chick oviduct.In previous papers we have described the purification to a,pparent homogeneity of calf thymus RNA polymerase A1 [I], BI and BII [2,3] and of rat liver RNA polymerase B activity 141. It has been observed that each enzyme is large and most probably composed of several polypeptide chains [l-61. Measurement of the size and of the stoichiometry of the subunits composing the different enzymes will provide This paper is part 9 of a series. Enzyme. RNA polymerase or nucleoside triphosphate RNA nucleotidyltransferase (EC 2.7.7.6). a basis for determ...

This paper describes a large-scale method for the solubilization and the separation of calf thymus A and B DNA-dependent RNA polymerase activities. This method is reproducible, gives high yields and can handle up to several kg of tissue. The solubilization method involved homogenization of the whole tissue, sonication a t high ionic strength, ammonium sulphate precipitation and protamine sulphate precipitation to remove nucleic acids. The separation of the two types of activities A (insensitive to a-amanitin) and B (inhibited by this compound) was achieved by differential adsorption to DEAE-cellulose using a batch procedure. Studies of the inhibitory effect of a-amanitin on the various fraction during the solubilization and the separation stages suggest very strongly that the two types of activities pre-existed in vivo and did not result from the solubilization procedure.The separation of several peaks of DNA-dependent RNA polymerase activities on DEAE-cellulose or DEAE-Sephadex columns [1,2] and the differential effect of the inhibitor a-amanitin on these peaks [2] suggested that animal cells contained multiple RNA polymerases with specific intranuclear localizations [3]. Definite proof of the existence of several distinct enzymes in animal cells requires the purification of these activities and the demonstration that they correspond to different proteins. To carry out such studies, for which milligram quantities of highly purified enzyme are required, it was necessary to work out a large-scale purification procedure since the content of DNAdependent RNA polymerase in animal tissues, on a weight basis, is at the most 'Ilo of that of procaryotic cells (see Conclusion). This paper describes the first steps of such a procedure, which can be applied to kilogram quantities of tissue and which results in the separation of two types of activity; type A which is insensitive to a-amanitin and type B which is completely inhibited a t very low concentrations of this compound. Preliminary reports of this work have been published [2,4].