Animal DNA-Dependent RNA Polymerases. Molecular Structures and Immunological Properties of Calf-Thymus Enzyme AI and of Calf-Thymus and Rat-Liver Enzymes B

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...

Animal DNA-Dependent RNA Polymerases. Purification and Molecular Structure of Hen-Oviduct and Liver Class-B RNA Polymerases

“…LIV, liver; OV. oviduct; CT, calf thymus SB3, SB4, SB5 and SB6, which have the same molecular weight as the calf thymus subunits of RNA polymerase BI and BII [5,6]. In addition, as previously found for calf thymus B enzymes [6] a minor band SB5' and components moving faster than SB6 are also seen.…”

“…At the present level of resolution the only difference between calf and hen polymerases B appears to lie in a slight difference of the charge of one subunit (SB2a) of enzyme form BIIa. This striking conservation of the molecular structure of RNA polymerases B is in keeping with their immunological properties [6,17] and reactivity with amatoxins, and indicates a high degree of preservation from mutational drift during evolution.…”

“…It is equally difficult to understand why three forms of RNA polymerase B (BO, BI and BIIb) would be required in rat liver [6] and only one in hen liver.…”

“…Finally, gel electrophoresis of the oviduct B enzymes in the presence of urea (Fig. 6 , gels 2 and 3) indicates that the SB2 component of hen oviduct RNA polymerase form BII is split into two bands, SB2a and SBZb, which correspond to the two bands SB2a and SB2b of the calf thymus enzyme forms BIIa and BIIb [6]. However, although the oviduct subunit SB2b has the same …”

“…Human y-globulins were purchased from Sigma (St. Louis, U.S.A.). Polyacrylamide gel electrophoresis under non-denaturating conditions (in the absence or in the presence of labelled [3H]amanitin) or under denaturating conditions (in the presence of sodium dodecylsulfate or urea) was carried out as previously reported [5,6,8]. RNA polymerase activity was measured as described before [5,7] in the presence of 50 mM (NH,),-SO,.…”