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A Unique Structure at the Carboxyl Terminus of the Largest Subunit of Eukaryotic RNA Polymerase II (Transcription/Gene Expression/Protein Phosphorylation) JEFFRY L

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A Unique Structure at the Carboxyl Terminus of the Largest Subunit of Eukaryotic RNA Polymerase II (Transcription/Gene Expression/Protein Phosphorylation) JEFFRY L Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7934-7938, December 1985 Biochemistry A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II (transcription/gene expression/protein phosphorylation) JEFFRY L. CORDEN*, DEBORAH L. CADENAt, JOSEPH M. AHEARN, JR.*, AND MICHAEL E. DAHMUSt *Howard Hughes Medical Institute Laboratory, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and tDepartment of Biochemistry and Biophysics, University of California, Davis, CA 95616 Communicated by Daniel Nathans, August 2, 1985 ABSTRACT Purified eukaryotic nuclear RNA polymerase alous mobility in NaDodSO4 gels due to postsynthetic mod- II consists of three subspecies that differ in the apparent ification cannot be ruled out. The three forms of the largest molecular masses of their largest subunit, designated Ho, Ha, subunit Ho (240 kDa), Iha (210-220 kDa), and lIb (170-180 and HIb for polymerase species HO, HA, and BIB, respectively. kDa) also differ in their ability to be phosphorylated both in Subunits Ho, Ha, and IHb are the products of a single gene. We vitro and in vivo (14). Subunit lIc (140 kDa) is structurally present here the amino acid composition of calf thymus different from IHo, Iha, and IIb (2, 33) and is present in all subunits Ha and lIb and the C-terminal amino acid sequence three forms of RNA polymerase II in equimolar stoichiom- of subunit Ha (Ho) inferred from the nucleotide sequence of etry with the largest subunit. part of the mouse gene encoding this RNA polymerase subunit. A monoclonal antibody prepared against calfthymus RNA The calculated amino acid composition ofthe peptide unique to polymerase II has been shown to recognize a determinant on subunit Ha indicates that subunit Ha contains a domain rich in subunit Iha (IIo) but not on IIb (15). This antibody does not serine, proline, threonine, and tyrosine. The sequence at the 3' inhibit nonspecific transcription on a nicked DNA template end of the mouse RNA polymerase H largest subunit gene but completely inhibits specific in vitro transcription from the reveals that the C-terminal domain consists of 52 repeats of a adenovirus 2 major-late promoter in a HeLa cell S100 extract seven amino acid block with the consensus sequence Tyr-Ser- (16). This result suggests a role for the domain retained in Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it subunit Iha in specific transcription. contains a high percentage of potential phosphorylation sites. In this paper we present the amino acid composition ofcalf thymus RNA polymerase II subunits Iha and Ilb. Comparison Eukaryotic nuclear RNA polymerases have been purified of the calculated amino acid composition of the peptide from a number of species and their complex subunit structure unique to subunit Iha with the DNA sequence at the 3' end of has been determined (1, 2). The three major classes, desig- the mouse RNA polymerase II large subunit gene reveals a nated I, II, and III, differ in subunit structure and transcrip- unique protein domain at the carboxyl terminus ofthe largest tive function. Each enzyme contains from 9 to 14 subunits subunit. and has a total molecular mass of 5-6 x 105. RNA polymerases I, II, and III each contain two nonidentical large MATERIALS AND METHODS subunits (>100 kDa) and 7-12 smaller subunits. The large subunits of one class are not shared by other classes but are Purification of RNA Polymerase H Subunits. RNA poly- highly conserved within each class among different species merase II was purified by a modification of the method of (3-6). Hodo and Blatti (17). Protein concentrations were estimated The only subunit that has been assigned a tentative by the method of Bradford (18). functional role is the largest subunit of RNA polymerase II. RNA polymerase II subunits were purified by a modifica- Greenleaf and collaborators have isolated a strain of tion of the method described by Hunkapiller et al. (19). Drosophila resistant to the polymerase II inhibitor a- Heparin-Sepharose fractions (up to 1.5 mg of RNA polymer- amanitin (7). This mutation (C4) was localized to band lOC on ase per gel) were loaded onto preparative 5% acrylamide gels the X chromosome and this region was cloned by P-element (1.5 mm in thickness). Sodium thioglycolate (0.1 mM) was tagging (8, 9). A 7-kilobase (kb) transcript from this region added to the upper reservoir buffer and gels were run as was shown to encode the largest subunit of RNA polymerase described by Laemmli (20). Protein bands were visualized by II (10). The Drosophila largest subunit gene has been cross- soaking the gels in 4 M sodium acetate for 5-10 min (21). The hybridized to a number ofeukaryotic DNAs (11) and has been bands were excised and placed in dialysis tubing (6.4 mm in used to clone the homologous genes from several species, diameter). The gel bands were placed in a Trans-Blot unit including yeast (12) and mouse (unpublished data). Sequence (Bio-Rad) and electroeluted at 600 mA (20 V) for 6 hr in 0.05 analyses of the mouse and Drosophila clones have shown M ammonium bicarbonate buffer containing 0.1% NaDod- that the genes are highly conserved and are homologous to SO4. The buffer temperature was maintained at 20-22°C. The the ' subunit of Escherichia coli RNA polymerase (unpub- eluant was removed from the dialysis tubing and samples lished data), implicating the largest subunit in the processes from a single gel were concentrated to .-0.5 ml with a of DNA binding and RNA chain elongation. Millipore ultrafiltration unit (immersible CX-10 filters) and Three different forms of RNA polymerase II have been lyophilized. The lyophilized sample was dissolved in 500 ,l described in a number of species. These enzymes, designated of water. Aliquots of 200 ,l were loaded onto 1-ml Sephadex IIO, IIA, and IIB, differ in the apparent molecular masses of G-25 columns equilibrated in water or 0.04% NaDodSO4 (22). their largest subunit (IIo, Ha, and IIb) and appear to be The columns were centrifuged in a clinical centrifuge and the related in part by limited proteolysis (13). However, anom- effluent was pooled. Samples were stored at -80°C. Polyacrylamide Gel Electrophoresis. NaDodSO4/polyacryl- The publication costs of this article were defrayed in part by page charge amide gel electrophoresis was carried out in slabs according payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: kb, kilobase(s); bp, base pair(s). 7934 Biochemistry: Corden et al. Proc. Natl. Acad. Sci. USA 82 (1985) 7935 to the method of Laemmli (20). The resolving gel was 5% Table 1. Amino acid composition of calf thymus RNA acrylamide. Gels were silver stained as described by Wray et polymerase subunits Ha and lIb and the predicted al. (23). C-terminal domains of calf and mouse subunit Ha Amino Acid Analysis. Aliquots containing 20 Ag of protein Calf thymus were lyophilized in hydrolysis tubes. Following the addition Calf thymus difference Mouset of 1 ml of constant-boiling HCl (Pierce) and 100 Al of 5% subunit,* peptide mol% phenol (wt/vol), the samples were hydrolyzed at 1100C for Amino mol% Ia - IIb,t (C-terminal 24, 48, or 72 hr. Performic acid oxidation was carried out as acid Ha Ilb mol% exon) described by Hirs (24) except that HBr was omitted. After hydrolysis, samples were dried under a stream ofnitrogen on Asx 9.2 11.1 2 a heating block at 1000C. Samples were either dissolved in 0.2 Thr 7.2 6.2 12 16 M lithium citrate buffer (pH 2.2) and analyzed on a Beckman Ser§ 10.4 5.5 36 33 6300 amino acid analyzer or dissolved in 0.2 M sodium citrate Glx 9.4 11.1 (pH 2.2) and analyzed on a Durram D-500 amino acid Pro 8.9 5.1 29 27 analyzer. Amino acid analyses were conducted at the Protein Gly 7.5 8.0 5 Structure Laboratory, University of California, Davis. Ala 5.5 5.8 4 1 General Nucleic Acid Methods. Details ofcloning, mapping, Vall 5.9 7.0 and sequencing the mouse RNA polymerase II gene will be Blel 4.7 5.6 published when the entire gene has been sequenced. Restric- Leu 7.5 9.3 tion enzyme digestions, blotting, and hybridization were Tyr 3.9 2.0 14 14 carried out by standard procedures (25). Sequencing was by Phe 2.9 3.4 the dideoxy chain-termination method (26) on subclones Lys 5.4 6.2 2 generated by unidirectional nuclease BAL-31 digestion. His 2.2 2.7 Arg 5.4 6.4 Cys1 1.2 1.2 1 RESULTS Met** 2.9 3.4 Trp Amino Acid Composition of Calf Thymus RNA Polymerase *Average of four determinations. I Subunits fIa and fIb. Calf thymus RNA polymerase II tCalculations were based on molecular weight estimates for Ila subunits were purified as described. The results, shown in (214,000) and Ilb (180,000) reported by Kedinger et al. (27) and are Fig. 1, indicate that there is no detectable contamination of expressed as mol% of the difference peptide. The precision of the the purified subunits with other subunits or proteins. amino acid analyzer is ±5%. Only those amino acids that differ Table 1 shows the amino acid composition of calf thymus signifcantly in amount between subunits Ha and Ilb were consid- ered in calculation of the mol% of the difference peptide. RNA polymerase subunits Ila and IIb.
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