Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides.

Abstract

RNA polymerase I was purified to homogeneity from Morris hepatoma 3924A. Purified RNA polymerase I contained a protein kinase activity that comigrated with the polymerase in nondenaturing gels. RNA polymerase I, purified from the same hepatoma, lacked protein kinase activity. Analysis of the subunit composition ofthe RNA polymerase I showed the presence of eight polypeptides: SI, Mr 190,000; S2, Mr 120,000; S3, Mr 62,000; S4, Mr 42,000; S5, Mr 24,600; S6, Mr 21,000; S7, Mr 19,500; and S8, Mr 17,500. Antibodies prepared against purified polymerase I specifically inhibited RNA synthesis catalyzed by RNA polymerase I. When subunits ofthe enzyme were covalently linked to diazobenzyloxymethyl paper, complexes between the antibody preparation and S1-S6 were visualized. No immune complexes were formed between RNA polymerase I antibodies and RNA polymerase II subunits. The antibody preparation was able to inhibit both the protein phosphorylation catalyzed by RNA polymerase I and that catalyzed by a nuclear kinase (NIl) purified from the same hepatoma. The two polypeptides of the nuclear kinaseMr 42,000 and 24,600 (identical in size to S4 and S5 of polymerase I-formed visible complexes with the RNA polymerase I antibodies. Both S4 and S5 of the polymerase contained an ATP binding site, a property associated with protein phosphorylation and also exhibited by the polypeptides of the purified kinase. These data suggest that polypeptides Of Mr 42,000 and 24,600 associated with polymerase I are responsible for its kinase activity.Phosphorylation of nuclear proteins has been correlated with enhanced gene expression in several systems (1). A number of observations suggest that ribosomal gene transcription may be regulated by protein phosphorylation (2-6). We have recently purified (6) a spermine-activated, cyclic nucleotide-independent nuclear protein kinase (classified as NII). This kinase has a high affinity for RNA polymerase I and stimulates RNA synthesis in vitro. We Preparation ofAntibodies. Male New Zealand White rabbits were injected biweekly with 50-100 ,ug of purified RNA polymerase I emulsified with an equal volume (0.5-1.0 ml) of Freund complete adjuvant. An IgG fraction was obtained from the sera (collected 8-20 weeks after the first injection) by (NH4)2SO4 precipitation (50% saturation at 4°C), followed by chromatography on Sephacryl S-200. IgG preparations containing traces of ribonuclease or protein phosphatase were further purified by affnity chromatography on protein A-Sepharose. Nonimmune IgG samples were prepared in the same manner from sera of uninjected rabbits.Analysis of Antibody Binding to Polypeptides on Diazobenzyloxymethyl-Paper. After polyacrylamide gel electrophoresis under denaturing conditions (8), the purified enzyme subunits were transferred to diazobenzyloxymethyl (DBM)-paper (9) by horizontal electrophoresis for 1 hr at 24 V. Complete (>90%) transfer of polypeptides to the DBM-paper was confirmed by staining gels after horizontal electrophoresis. After transfer, the DBM-paper was in...

Immunocytochemical localization of RNA polymerase I in the fibrillar centers of nucleoli

“…template-engaged RP I also seems unlikely since the rabbit antibodies used contain IgGs directed against epitopes of most of the RP I-subunits (Rose et al, 1981).…”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…The antibodies used were raised in rabbits against purified RNA polymerase I from rat Morris hepatoma (Rose et al, 1981) or were obtained from sera of patients suffering from scleroderma auto immune disease (Reimer et al,I 987a,b). Immunofluorescence microscopy clearly reveals that these antibodies specifically and intensely react with the nucleoli of mammalian cells such as cultured rat cells (Fig.…”

Identification and Definition of nucleolus-related fibrillar bodies in micronucleated cells

“…The following antibodies were used: (a) Rabbit antibodies against RNA polymerase I from rat hepatoma [21,22], (b) human autoimmune serum (SI8) against RNA polymerase I [17], (c) murine monoclonal antibody 72B9 against a Mr -37,000 protein of the dense fibrillar component related to the U3 RNP complex [18], (d) human autoimmune serum (Scl C) recognizing a protein of the dense fibrillar component identified as fibrillarin and probably related to the antigen of monoclonal antibody 72B9 [18,19], (e) murine monoclonal antibody No-1I4 against a structural protein (Mr -180 ,000) of the dense fibrillar component of Xenopus nucleoli [23], (f) murine monoclonal antibody RSI-105 recognizing ribosomal protein SI located in the granular component [12] and (g) murine monoclonal antibody No-185 against the nonribosomal protein N038 , a member of the nucleoplasmin family and homologous to the mammalian protein B23 (for review see [2]) which is localized in the granular component of Xenopus nucleoli [24] . The immunofluorescence staining patterns of interphase and mitotic cells obtained with these antibodies are summarized in Table I.…”