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• W2068999898 abstract "Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the α subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic α subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II. Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the α subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic α subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II. Eukaryotes contain three classes of nuclear RNA polymerase, referred to as RNA polymerases I or A, II or B, and III or C. Each class of RNA polymerase is a multimeric enzyme composed of two unique large subunits in excess of 100 kDa that are related to β′ and β subunits of Escherichia coli RNA polymerase and 10 or more smaller subunits (reviewed in (1.Sentenac A.C. CRC Crit. Rev. Biochem. 1985; 18: 51-90Google Scholar, 2.Sentenac A. Riva M. Thuriaux P. Buhler J.M. Treich I. Carles C. Werner M. Ruet A. Huet J. Mann C. Chiannikulchai N. Stettler S. Mariotte S. Yamamoto K.R. McKnight S.L. Transcriptional Regulation. 1. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 27-54Google Scholar, 3.Thuriaux P. Sentenac A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of Yeast Saccharomyces: Gene Expression. II. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 1-48Google Scholar, 4.Woychik N.A. Young R.A. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York, NY1994: 227-242Google Scholar, 5.Young R.A. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (372) Google Scholar)). In the yeast Saccharomyces cerevisiae five of these smaller subunits are common to RNA polymerase I, II, and III, and seven subunits are common to RNA polymerases I and III(3.Thuriaux P. Sentenac A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of Yeast Saccharomyces: Gene Expression. II. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 1-48Google Scholar, 4.Woychik N.A. Young R.A. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York, NY1994: 227-242Google Scholar). Subunits of about 40 kDa (e.g. yeast AC40 and B44 or RPB3) and 12.5-19 kDa (e.g. yeast AC19 and B12.5 or RPB11) in RNA polymerase I, II, and III have limited amino acid sequence homology with the α subunit of the prokaryotic RNA polymerase(6.Dequard-Chablat M. Riva M. Carles C. Sentenac A. J. Biol. Chem. 1991; 266: 15300-15307Abstract Full Text PDF PubMed Google Scholar, 7.Martindale D.W. Nucleic Acids Res. 1990; 18: 1953-1960Crossref Scopus (43) Google Scholar, 8.Woychik N.A. McKune K. Lane W.S. Young R.A. Gene Expr. 1993; 3: 77-82PubMed Google Scholar). The localized amino acid sequence homology between the eukaryotic α-like subunits and the α subunit in prokaryotic RNA polymerases has been referred to as the “α motif”(2.Sentenac A. Riva M. Thuriaux P. Buhler J.M. Treich I. Carles C. Werner M. Ruet A. Huet J. Mann C. Chiannikulchai N. Stettler S. Mariotte S. Yamamoto K.R. McKnight S.L. Transcriptional Regulation. 1. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 27-54Google Scholar, 9.Lalo D. Carles C. Sentenac A. Thuriaux P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5524-5528Crossref PubMed Scopus (109) Google Scholar). The yeast AC40 and AC19 subunits are common to RNA polymerase I and III, and the related subunits, B44 or RPB3 and B12.5 or RPB11, are unique to RNA polymerase II(6.Dequard-Chablat M. Riva M. Carles C. Sentenac A. J. Biol. Chem. 1991; 266: 15300-15307Abstract Full Text PDF PubMed Google Scholar, 8.Woychik N.A. McKune K. Lane W.S. Young R.A. Gene Expr. 1993; 3: 77-82PubMed Google Scholar, 10.Kolodziej P. Young R.A. Mol. Cell. Biol. 1989; 9: 5387-5394Crossref PubMed Scopus (67) Google Scholar, 11.Mann C. Buhler J.-M. Treich I. Sentenac A. Cell. 1987; 48: 627-637Abstract Full Text PDF PubMed Scopus (111) Google Scholar). Bacterial RNA polymerase has an α subunit with a stoichiometry of 2, and the core enzyme is composed of α2ββ′(12.Burgess R.R. J. Biol. Chem. 1969; 244: 6168-6176Abstract Full Text PDF PubMed Google Scholar). The yeast B44 subunit is reported to have a stoichiometry of 2 in RNA polymerase II(13.Kolodziej P. Woychik N. Liao S.M. Young R.A. Mol. Cell. Biol. 1990; 10: 1915-1920Crossref PubMed Scopus (101) Google Scholar), but the AC40 and AC19 subunits in RNA polymerases I and III have apparent stoichiometries of 1(3.Thuriaux P. Sentenac A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of Yeast Saccharomyces: Gene Expression. II. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 1-48Google Scholar, 14.Huet J. Riva M. Sentenac A. Fromageot P. J. Biol. Chem. 1985; 260: 15304-15310Abstract Full Text PDF PubMed Google Scholar). The stoichiometry of the yeast B12.5 subunit has not been reported. Yeast RNA polymerase II contains a total of 12 subunits, and each of these is encoded by a single copy gene (reviewed in (3.Thuriaux P. Sentenac A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of Yeast Saccharomyces: Gene Expression. II. Cold Spring Harbor Laboratory Press, Plainview, NY1992: 1-48Google Scholar) and (4.Woychik N.A. Young R.A. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York, NY1994: 227-242Google Scholar)). All of the RNA polymerase II subunit genes in yeast have been sequenced. Only a limited number of RNA polymerase II subunit genes in other eukaryotes have been cloned and sequenced(4.Woychik N.A. Young R.A. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York, NY1994: 227-242Google Scholar). With the exception of genes encoding the largest subunit of RNA polymerase II in soybean and trypanosomes(15.Dietrich M.A. Prenger J.P. Guilfoyle T.J. Plant Mol. Biol. 1990; 15: 207-223Crossref PubMed Scopus (33) Google Scholar, 16.Evers R. Hammer A. Kock J. Jess W. Borst P. Memet S. Cornelissen A.W.C.A. Cell. 1989; 56: 585-597Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 17.Smith J.L. Levin J.R. Ingles C.J. Agabian N. Cell. 1989; 56: 815-827Abstract Full Text PDF PubMed Scopus (69) Google Scholar), those RNA polymerase II subunit genes that have been identified in organisms besides yeast are reported to be single copy genes. Nuclear RNA polymerase subunit-subunit interactions, subunit functions, and assembly pathways are only beginning to be unraveled. For example, the AC40 and AC19 subunits of yeast RNA polymerase I and III have been shown to associate with one another in a yeast two-hybrid system(9.Lalo D. Carles C. Sentenac A. Thuriaux P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5524-5528Crossref PubMed Scopus (109) Google Scholar). Extragenic suppression of mutations in the AC40 and AC19 subunit genes confirmed the interaction between these two subunits and a third subunit, ABC10β(9.Lalo D. Carles C. Sentenac A. Thuriaux P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5524-5528Crossref PubMed Scopus (109) Google Scholar). Studies on mutations in the three largest subunits of yeast RNA polymerase II indicate that the B44 subunit associates with second largest subunit (i.e. B150 or RPB2), which in turn complexes with the largest subunit (i.e. B220 or RPB1) to facilitate assembly of the enzyme(18.Kolodziej P. Young R.A. Mol. Cell. Biol. 1991; 11: 4669-4678Crossref PubMed Scopus (55) Google Scholar). Here, we report on the cloning and sequencing of genes and/or cDNAs for the 36-kDa (B36a and B36b) and 13.6-kDa (B13.6) subunits in Arabidopsis RNA polymerase II, which are homologs to yeast B44 and B12.5 (i.e. encoded by the RPB3 and RPB11 genes in S. cerevisiae), respectively, to determine the stoichiometry of the B36 subunit in the enzyme and investigate its self-association and its association with the B13.6 subunit. An Arabidopsis thaliana (ecotype Columbia) cDNA library in λYES (19.Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (322) Google Scholar) was used for antibody screening. Approximately 5 × 105 plaque-forming units were plated on an E. coli Y1090 lawn at moderate density and blotted onto Immobilon-NC (HATF) membranes (Millipore, Bedford, MA). Replica filters were probed with affinity-purified antibody (20.Ulmasov T. Guilfoyle T. J. Biol. Chem. 1992; 267: 23165-23169Abstract Full Text PDF PubMed Google Scholar, 21.Guilfoyle T.J. Hagen G. Malcolm S. J. Biol. Chem. 1984; 259: 640-648Abstract Full Text PDF PubMed Google Scholar) raised against the 40-kDa subunit of cauliflower (Brassica oleraceae) RNA polymerase II (60 ng of IgG/ml in Tris-buffered saline and 1% nonfat dry milk) at room temperature for 24 h. Filters were washed with three changes of Tris-buffered saline and then reacted with goat anti-rabbit IgG conjugated to alkaline phosphatase for 90 min at room temperature. Secondary antibody and reaction with alkaline phosphatase were carried out according to the supplier's instructions (5 Prime → 3 Prime, Boulder, CO). The AtRPB36a cDNA clone insert was used to screen another 5 × 105 plaque-forming units of the λYES cDNA library(19.Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (322) Google Scholar). Six positive clones were selected, purified, and sequenced. Five of the six clones contained identical sequence (of varying length) to the AtRPB36a cDNA clone, and the remaining clone, AtRPB36b, contained a similar but distinct sequence. An EST cDNA clone (GenBank(™) accession number Z47635) from an Arabidopsis cell suspension library (22.Regad F. Bardet C. Tremousaygue D. Moisan A. Lescure B. Axelos M. FEBS Lett. 1993; 316: 133-136Crossref PubMed Scopus (96) Google Scholar) was identified which had homology to yeast RPB11(8.Woychik N.A. McKune K. Lane W.S. Young R.A. Gene Expr. 1993; 3: 77-82PubMed Google Scholar). The EST sequence was reported as a partial sequence of a full-length cDNA clone. The complete sequence of this cDNA clone was obtained from the EST cDNA clone, which was provided by Dr. Gabriel Phillips (Laboratoire de Biologie Moleculaire de Plantes, CNRS, Strasbourg Cedex, France). We refer to this clone as AtRPB13.6. Both AtRPB36a and AtRPB36b cDNA clone inserts were used to screen an A. thaliana (ecotype Columbia) λEMBL3 genomic library (provided by Harry Klee, Monsanto Chemical Company, St. Louis, MO). Genomic clones were selected, purified, and mapped with restriction enzymes. Restriction fragments corresponding to genomic fragments of AtRPB36a and AtRPB36b were subcloned into pBluescript (Stratagene, La Jolla, CA) vectors or pMOB (23.Strathmann M. Hamilton B.A. Mayeda C.A. Simon M.I. Meyerowitz E.M. Palazzolo M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1247-1250Crossref PubMed Scopus (178) Google Scholar) and sequenced using a Tn1000 kit (Gold Biotechnology, St. Louis, MO). Oligonucleotides used for sequencing and cloning procedures were synthesized at the University of Missouri DNA Core Facility. Sequencing was performed manually using Sequenase (U.S Biochemical Corp.) and by automated sequencing using the DyeDeoxy(™) procedure (Applied BioSystems Inc., Foster City, CA). Computer analysis was performed using the BLAST family of programs (24.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) and the E-mail BLAST server at National Center for Biotechnology Information, the Genetics Computer Group package (Genetics Computer Program, Madison, WI), and IBM Pustell Sequence Analysis software (International Biotechnologies, Inc., New Haven, CT). Protein sequence alignments were done using GAP, BESTFIT, and PILEUP programs from the GCG package with the Gap Weight and Gap Length Weight parameters 3.0 and 0.1, respectively. A. thaliana (ecotype Columbia) genomic DNA was isolated, treated with restriction enzymes, and blotted onto nylon membranes using standard methods(20.Ulmasov T. Guilfoyle T. J. Biol. Chem. 1992; 267: 23165-23169Abstract Full Text PDF PubMed Google Scholar, 25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/John Wiley and Sons, Inc., New York1995Google Scholar, 26.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Hybridization was carried out in 6 × SSC (1 × SSC is 0.15 M NaCl + 0.015 sodium citrate) containing 1% SDS and 100 μg/ml heparin at 60°C, and washings were conducted in 2 × SSC at 55°C. Northern blotting was carried out with 2 μg of poly(A)+ RNA isolated from Arabidopsis suspension culture cells(27.Doelling J.H. Pikaard C.S. Plant Cell Rep. 1993; 12: 241-244Crossref PubMed Scopus (48) Google Scholar). RNA was isolated by a standard protocol(25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/John Wiley and Sons, Inc., New York1995Google Scholar), denatured with glyoxal and Me2SO, subjected to electrophoresis on 1.4% agarose gels, and transferred to a nylon membrane(26.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). AtRPB36a and AtRPB36b cDNAs were labeled with 32P using the Prime-a-Gene labeling system (Promega Corp., Madison, WI). A mixed probe was used for hybridization in 6 × SSPE(26.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), 1% nonfat dry milk, 1% SDS, and 0.5 mg/ml denatured herring sperm DNA at 68°C. Washings were in 2 × SSC and 0.1% SDS for 15 min at 25°C, 0.5 × SSC and 0.1% SDS for 15 min at 25°C, and 0.2 × SSC and 1% SDS for 30 min at 50°C Small N-terminal portions of open reading frames of AtRPB36a and AtRPB36b were isolated using specific primers, Pfu DNA polymerase (Stratagene), and polymerase chain reaction. These were cloned in-frame to the HisTag(™) pET-16b expression vector (Novagen, Madison, WI). In order to avoid mistakes introduced by polymerase chain reaction, most of the ORFs ( 1The abbreviations used are: ORFopen reading frameHAhemagglutininkbkilobase pair(s).) (with the exception of small N-terminal regions) were cloned by using corresponding restriction fragments from the original cDNAs. The N-terminal regions made by polymerase chain reaction were verified by sequencing. Expression of the clones was induced by the addition of IPTG to midlog cultures of the BL21(DE3) strain of E. coli. After 2-3 h of induction, cells were harvested and sonicated. Fusion proteins were purified from inclusion bodies under denaturing conditions as described by the supplier (Novagen). open reading frame hemagglutinin kilobase pair(s). ORFs from AtRPB36a, AtRPB36b, and AtRPB13.6 cDNAs were fused to the T3 phage promoter in a pBluescript II KS+ or SK+ vectors (Stratagene) with a TMV Ω translational enhancer(28.Gallie D.R. Sleat D.E. Watts J.W. Turner P.C. Wilson T.M.A. Nucleic Acids Res. 1987; 15: 3257-3273Crossref PubMed Scopus (322) Google Scholar). RNA was synthesized using the RiboMax(™) T3 system (Promega, Madison, WI). One-μg aliquots of in vitro synthesized RNA were used to program a FlexiRabbit(™) rabbit reticulocyte in vitro translation system (Promega, Madison, WI) with [35S]methionine (DuPont NEN). Translation products were monitored on 10% SDS-polyacrylamide gels(29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), 10% Tricine-SDS(30.Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar), or nondenaturing 7.5% polyacrylamide gels and subjected to autoradiography. Nondenaturing gels were constructed and subjected to electrophoresis at 4°C using the Laemmli system (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) minus SDS. A double-stranded oligonucleotide was cloned upstream of the start codon for a given ORF to create an in-frame influenza hemagglutinin (HA) epitope tag. These constructs encoded proteins with a 12-amino acid extension, MGYPYDVPDYAH (the HA epitope is underlined), at their N terminus. Epitope-tagged and untagged cDNAs were co-translated in vitro (as described above), and the 35S-labeled in vitro translation products were immunoprecipitated with 12CA5 monoclonal antibodies (Berkeley Antibody Company, Richmond, CA), which were immobilized on Protein A-agarose (Sigma). Five μl of immobilized antibody was added to 10 μl of in vitro translation mixture and adjusted to 200 μl with PBS/Tween (phosphate-buffered saline containing 0.05% Tween 20). Immunoprecipitation was carried out at 4°C for 12 h in a rotator. After incubation, the resin was washed five times with 1 ml of ice-cold PBS/Tween, and immunoprecipitates were eluted from the resin in 50 μl of SDS sample buffer(29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Five μl samples were resolved on 10 or 15% SDS gels and subjected to autoradiography. A full-length AtRPB13.6 ORF was cloned in-frame with a GAL4 DNA-binding domain into a pAS1 vector, and full-length AtRPB36a and AtRPB36b ORFs were cloned in-frame with the GAL4 activation domain into pACTII(31.Durfee T. Becherer K. Chen P-L. Yeh S-H. Yang Y. Kilburn A.P. Lee W-H. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). Both plasmids were sequentially introduced into yeast strain JC981 (an Ade+ derivative of Y187; obtained from John Cannon, University of Missouri; (31.Durfee T. Becherer K. Chen P-L. Yeh S-H. Yang Y. Kilburn A.P. Lee W-H. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar)). Transformed yeast cells were grown in Trp(-) Leu(-)/sucrose complete medium(25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/John Wiley and Sons, Inc., New York1995Google Scholar). Mid-log cultures were harvested, and β-galactosidase activity was assayed by a nitrocellulose filter/liquid N2/5-bromo-4-chloro-3-indoyl β-D-galactoside method or assayed directly in lysates as described by Ausubel et al.(25.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/John Wiley and Sons, Inc., New York1995Google Scholar) using a chemiluminescent assay, Galacto-Light(™) (Tropix, Bedford, MA). Truncations in the N-terminal or C-terminal portions of the B36b subunit were made using a series of restriction endonuclease sites. The B36b subunit was chosen for truncations because of some convenient restriction endonuclease sites not found in the B36a subunit cDNA. A chimeric construct was made that consisted of amino acids 249-319 of the B36b subunit fused to the C terminus of GH2/4(32.Hagen G. Uhrhammer N. Guilfoyle T.J. J. Biol. Chem. 1988; 263: 6442-6446Abstract Full Text PDF PubMed Google Scholar). The GH2/4 cDNA encodes a glutathione S-transferase(33.Ulmasov T. Ohmiya A. Hagen G. Guilfoyle T. Plant Physiol. 1995; 108: 919-927Crossref PubMed Scopus (87) Google Scholar). A. thaliana (ecotype Columbia) cell cultures were grown in liquid media (27.Doelling J.H. Pikaard C.S. Plant Cell Rep. 1993; 12: 241-244Crossref PubMed Scopus (48) Google Scholar) as 1-liter cultures in 4-liter flasks with constant agitation on a rotatory shaker at 25°C. Eight days after subculture, cells were harvested on two layers of Miracloth (Calbiochem, La Jolla, CA) using a Buchner funnel. Cells were washed with several volumes of cold distilled H20. Water was removed by vacuum filtration, and cells were frozen in liquid N2 and stored at −80°C prior to RNA polymerase II purification. For purification of RNA polymerase II, 200 g of cells were thawed and suspended in 200 ml of grinding buffer (50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 60 mM ammonium sulfate, 0.5 mM dithiothreitol, and 20% (v/v) ethylene glycol) containing 1 mM Pefabloc SM (Boehringer Mannheim) 10 μg/ml aprotinin, 1 μg/ml pepstatin, 300 μg/ml benzamidine, and 10 μg/ml leupeptin. All purification steps were carried out at 4°C. Cells were broken by grinding for 2 min using full speed with a Polytron PT20ST and subsequently with 15 30-s bursts and 90-s intermittent periods with a Bead-Beater and 100 g of acid-washed glass beads (425-600 microns; Sigma). The homogenate was filtered through two layers of Miracloth and centrifuged at 10,000 × g for 20 min. The supernatant was collected, and RNA polymerase II was purified by precipitation with and elution from Polymin P, ammonium sulfate precipitation, and chromatography on DEAE cellulose and phosphocellulose as described by Jendrisak and Burgess(34.Jendrisak J.J. Burgess R.R. Biochemistry. 1975; 14: 4639-4645Crossref PubMed Scopus (193) Google Scholar). The phosphocellulose fraction was dialyzed against 20 mM HEPES (pH 7.8), 0.1 mM EDTA, 0.5 mM dithiothreitol, and 50% glycerol, and dialysate was stored frozen at −80°C. Wheat germ RNA polymerase II was purified using the methods of Jendrisak and Burgess (34.Jendrisak J.J. Burgess R.R. Biochemistry. 1975; 14: 4639-4645Crossref PubMed Scopus (193) Google Scholar) with final chromatography on heparin-Sepharose(21.Guilfoyle T.J. Hagen G. Malcolm S. J. Biol. Chem. 1984; 259: 640-648Abstract Full Text PDF PubMed Google Scholar). The purified RNA polymerase II was judged to be nearly homogeneous on 15% SDS-polyacrylamide gels (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) when compared with purified wheat germ RNA polymerase II. Subunit stoichiometries were determined for the three largest subunits (205 + 175-, 135-, and 36-kDa subunits) in purified Arabidopsis RNA polymerase II using 7.5 and 15% SDS gels. Peak areas for the three largest subunits were measured for Coomassie Blue-stained gels using Image I Software (Universal Image, Corp., Westchester, PA). Quantitation of 35S incorporation into the three largest subunits was carried out with a Fuji BAS1000 instrument and MacBAS1000 software (Fuji Medical Systems, Stamford, CT). The nucleotide sequences for the AtRPBC13.6 cDNA clone and the AtRPB36a and AtRPB36b genes reported in this paper are entered in the GenBank(™) nucleotide sequence data base with accession numbers U28048, L34770, and L34771, respectively. The accession number for the Arabidopsis AC14 EST cDNA clone is Z25617. A polyclonal antibody raised against the 40-kDa subunit of cauliflower RNA polymerase II (21.Guilfoyle T.J. Hagen G. Malcolm S. J. Biol. Chem. 1984; 259: 640-648Abstract Full Text PDF PubMed Google Scholar) was affinity-purified on a wheat germ RNA polymerase II Affi-Gel resin (see “Materials and Methods” and (20.Ulmasov T. Guilfoyle T. J. Biol. Chem. 1992; 267: 23165-23169Abstract Full Text PDF PubMed Google Scholar)). The affinity purified antibody was used to screen an Arabidopsis cDNA expression library as described previously(20.Ulmasov T. Guilfoyle T. J. Biol. Chem. 1992; 267: 23165-23169Abstract Full Text PDF PubMed Google Scholar). Seventeen positive clones were selected from primary screening, and five were purified to homogeneity. One of the purified clones, AtRPB36a, was sequenced and found to be related in amino acid sequence to yeast RPB3 (10.Kolodziej P. Young R.A. Mol. Cell. Biol. 1989; 9: 5387-5394Crossref PubMed Scopus (67) Google Scholar) and human hRPB33 ((35.Pati U.K. Weissman S.M. J. Biol. Chem. 1990; 265: 8400-8403Abstract Full Text PDF PubMed Google Scholar); Fig. 1). The Arabidopsis cDNA had an insert of 1.3 kb that contained an ORF encoding 319 amino acids with a predicted molecular mass of 35.5 kDa and an estimated pI of 4.4. The Arabidopsis B36a amino acid sequence showed 39% identity to the RPB3 subunit in yeast RNA polymerase II and 44% identity to the hRPB33 subunit in human RNA polymerase II but only 31% identity to the AC40 subunit in yeast RNA polymerases I and III(11.Mann C. Buhler J.-M. Treich I. Sentenac A. Cell. 1987; 48: 627-637Abstract Full Text PDF PubMed Scopus (111) Google Scholar). Southern blot analysis of Arabidopsis genomic DNA suggested that more than one copy of this subunit gene was present in the Arabidopsis genome because a variety of restriction endonucleases produced multiple restriction fragments (of varying intensities) that hybridized to the AtRPB36a cDNA probe (Fig. 2A). To determine if more than one gene encoded the 36-kDa RNA polymerase II subunit, we rescreened 5 × 105 plaque-forming units of the λYES cDNA library with the AtRPB36a cDNA and selected six positive clones. Each purified clone was partially sequenced. Five of these were identical in sequence to AtRPB36a with the exception of the position of the poly(A) tail in the 3′-untranslated region (data not shown), reflecting heterogeneity in the site selection for poly(A) addition. One of the clones contained a full-length cDNA that was related, but distinct from AtRPB36a. This 1.2-kb cDNA clone, AtRPB36b, contained an ORF encoding 319 amino acids with 88% identity to the amino acid sequence in AtRPB36a and 37% identical to yeast RPB3 ( Fig. 1). The predicted pI of the B36b protein was 4.7. Within the ORFs, AtRPB36a and AtRPB36b showed 91% identity in nucleotide sequence, and in the untranslated regions, the two cDNA clones were 82% identical (data not shown). A Northern blot with a mixed AtRPB36a and AtRPB36b probe revealed only one size mRNA of 1.5 kb ( Fig. 2B). We have not attempted to quantitate the relative amounts of the individual AtRPB36a and AtRPB36b mRNAs. The ORFs in AtRPB36a and AtRPB36b encode putative metal-binding motifs (i.e. “zinc-fingers”), CX2CX5CX2C, starting at position Cys90 in B36a (Fig. 1). The motif in B36b differs from that in B36a because the B36b clone contains an N-terminal extension of this motif, CX2CX2CX5CX2C. These putative metal-binding motifs differ slightly from those found in the homologous RNA polymerase II subunit in S. cerevisiae(10.Kolodziej P. Young R.A. Mol. Cell. Biol. 1989; 9: 5387-5394Crossref PubMed Scopus (67) Google Scholar), Schizosaccharomyces pombe(36.Azuma Y. Yamagishi M. Ishihama A. Nucleic Acids Res. 1993; 16: 3749-3754Crossref Scopus (44) Google Scholar), human(35.Pati U.K. Weissman S.M. J. Biol. Chem. 1990; 265: 8400-8403Abstract Full Text PDF PubMed Google Scholar), and Tetrahymena thermophila(7.Martindale D.W. Nucleic Acids Res. 1990; 18: 1953-1960Crossref Scopus (43) Google Scholar), which are conserved as CXCX5CX2C. The yeast RPB3 subunit has been reported to bind 65Zn using a zinc-blotting technique(37.Treich I. Riva M. Sentenac A. J. Biol. Chem. 1991; 266: 21971-21976Abstract Full Text PDF PubMed Google Scholar), and we have preliminary evidence that the Arabidopsis B36a and B36b subunits bind zinc using the methods of Treich et al.(37.Treich I. Riva M. Sentenac A. J. Biol. Chem. 1991; 266: 21971-21976Abstract Full Text PDF PubMed Google Scholar). ( 2T. Guilfoyle and T. Ulmasov, unpublished results.) The B36a and B36b subunits contain two motifs related to the prokaryotic RNA polymerase α subunit (Fig. 1). One of these motifs (the more N-terminal) consists of a stretch of amino acids that is referred to as the “α motif” ((7.Martindale D.W. Nucleic Acids Res. 1990; 18: 1953-1960Crossref Scopus (43) Google Scholar) and (9.Lalo D. Carles C. Sentenac A. Thuriaux P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5524-5528Crossref PubMed Scopus (109) Google Scholar); reviewed in Refs. 3 and 4). The second α-like motif consists of a leucine-rich C-terminal region including amino acids Leu" @default.
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