['Assembly', 'of', 'Transcriptionally', 'Active', 'RNA', 'Polymerase', '|', 'Initiation', 'Factor', 'SL1', 'from', 'Recombinant', 'Subunits', 'Joost', 'C.', 'B.', 'M.', 'Zomerdijk,', 'Holger', 'Beckmann,', 'Lucio', 'Comai,', 'Robert', 'Tjian*', 'Initiation', 'of', 'ribosomal', 'RNA', 'synthesis', 'by', 'RNA', 'polymerase', '|', 'requires', 'the', 'promoter', 'selec-', 'tivity', 'factor', 'SL1,', 'which', 'consists', 'of', 'the', 'TATA-binding', 'protein,', 'TBP,', 'and', 'three', 'associated', 'factors,', 'TAF,s', '110,', '63,', 'and', '48.', 'Here', 'the', 'in', 'vivo', 'and', 'in', 'vitro', 'assembly', 'of', 'functional', 'SL1', 'complexes', 'from', 'recombinant', 'TAF,s', 'and', 'TBP', 'are', 'reported.', 'Complexes', 'containing', 'TBP', 'and', 'alt', 'three', 'TAF,s', 'were', 'as', 'active', 'in', 'supporting', 'transcription', 'from', 'the', 'human', 'ribosomal', 'RNA', 'gene', 'promoter', 'as', 'endogenous', 'SL1,', 'whereas', 'partial', 'complexes', 'without', 'TBP', 'did', 'not', 'efficiently', 'direct', 'transcription', 'in', 'vitro.', 'These', 'results', 'suggest', 'that', 'TAF,s', '110,', '63,', 'and', '48,', 'together', 'with', 'TBP,', 'are', 'necessary', 'and', 'sufficient', 'to', 'reconstitute', 'a', 'transcriptionally', 'active', 'SL1', 'complex.', ' ', 'Transcription', 'of', 'the', 'genes', 'for', '28S', 'and', '185', 'ribosomal', 'RNA', 'by', 'RNA', 'polymerase', '|', '(RNA', 'pol', 'I)', 'in', 'mammalian', 'cells', 'is', 'a', 'highly', 'regulated', 'process', 'that', 'has', 'been', 'intensively', 'studied', 'for', 'about', 'two', 'decades', '(1).', 'Three', 'components,', 'RNA', 'pol', 'I,', 'the', 'upstream', 'bind-', 'ing', 'factor', 'UBF,', 'and', 'the', 'promoter', 'selectivity', 'factor', 'SLI', 'are', 'minimally', 'required', 'for', 'accu-', 'rate', 'initiation', 'of', 'transcription', 'from', 'the', 'hu-', 'man', 'ribosomal', 'promoter', '(2).', 'UBF', 'has', 'been', 'purified', 'to', 'homogeneity', 'and', 'cloned,', 'and', 'recombinant', 'versions', 'of', 'UBF', 'are', 'available', '(3).', 'Until', 'recently', 'the', 'subunit', 'composition', 'and', 'biochemical', 'activities', 'of', 'SL1', 'were', 'un-', 'known.', 'Recent', 'advances', 'in', 'antibody', 'affinity', 'chromatography', 'allowed', 'us', 'to', 'purify', 'small', 'quantities', 'of', 'SL}', 'from', 'HeLa', '(human)', 'nu-', 'clear', 'extracts', 'to', 'apparent', 'homogeneity.', 'These', 'studies', 'revealed', 'that', 'SL1', 'is', 'composed', 'of', 'the', 'TATA', 'binding', 'protein,', 'TBP,', 'and', 'at', 'least', 'three', 'associated', 'subunits', 'of', '110,', '63,', ' ', 'Howard', 'Hughes', 'Medical', 'Institute,', 'Oepartment', 'of', 'Molec-', 'ular', 'and', 'Cell', 'Biology,', 'University', 'of', 'Califomia,', 'Berkeley,', 'CA', '94720-3204,', 'USA.', '“To', 'whom', 'correspondence', 'should', 'be', 'addressed.', 'and', '48', 'kD,', 'called', 'TAF\\s', '(TBP', 'associated', 'factors', 'for', 'RNA', 'pol', 'I)', '(4).', 'Although', 'reconstitution', 'of', 'SL1', 'with', 're-', 'combinant', 'TBP', 'and', 'partially', 'purified', 'TAF,', 'complexes', 'that', 'contain', 'the', 'three', 'subunits', 'obtained', 'by', 'TBP', 'antibody', 'affinity', 'chroma-', 'tography', 'suggested', 'thar', 'these', 'associated', 'fac-', 'tors', 'are', 'important', 'for', 'the', 'assembly', 'of', 'tran-', 'scriptionally', 'active', 'SL1', 'complexes', '(4),', 'it', 'was', 'not', 'possible', 'to', 'determine', 'whether', 'these', 'subunits', 'were', 'sufficient', 'to', 'direct', 'promoter', 'and', 'RNA', 'polymerase', 'selectivity.', 'Further', 'pu-', 'tification', 'and', 'disruption', 'of', 'the', 'complex', 'and', 'the', 'reassembly', 'of', 'active', 'SLI', 'proved', 'to', 'be', 'impractical', 'given', 'the', 'low', 'amount', 'of', 'SL1', 'in', 'the', 'cell', 'and', 'the', 'limited', 'amounts', 'of', 'purified', 'material', 'obtainable.', 'Thus,', 'it', 'became', 'evident', 'that', 'future', 'advances', 'in', 'determining', 'the', 'structure', 'and', 'function', 'of', 'this', 'essential', 'hu-', 'man', 'transcription', 'complex', 'would', 'require', 'molecular', 'cloning', 'and', 'high', 'level', 'expression', 'of', 'each', 'subunit.', 'In', 'the', 'accompanying', 'paper,', 'we', 'describe', 'the', 'isolation', 'of', 'complementary', 'DNAs', '(cDNAs)', 'encoding', 'each', 'of', 'the', 'three', 'human', 'TAFjs', 'present', 'in', 'SLI', '(5).', 'Here,', 'we', 'describe', 'the', 'assembly', 'of', 'partial', 'and', 'com-', 'SCIENCE', '*', 'VOL.', '266', '*', '23', 'DECEMBER', '1994', 'plete', 'SLI', 'complexes.', 'We', 'have', 'assented', 'transcriptionally', 'active', 'SLE', 'in', 'HeLa', 'cells', 'infected', 'with', 'recombinant', 'vaccinia', 'viruses', 'expressing', 'TAF\\s', 'and', 'TBP.', 'As', 'an', 'alterna-', 'tive', 'procedure', 'for', 'reconstituting', 'SL1,', 'we', 'have', 'also', 'reconstructed', 'stable', 'complexes', 'containing', 'the', 'three', 'TAF,s', 'and', 'TBP', 'in', 'vitro', 'using', 'purified', 'recombinant', 'proteins.', 'After', 'isolating', 'these', 'in', 'vivo', 'and', 'in', 'vitro', 'assembled', 'complexes', 'by', 'antibody', 'affinity', 'chroma-', 'tography,', 'their', 'ability', 'to', 'support', 'accurace', 'initiation', 'of', 'transcription', 'from', 'the', 'human', 'ribosomal', 'promoter', 'in', 'a', 'reconstituted', 're-', 'action', 'containing', 'UBF', 'and', 'RNA', 'pol', '!', 'was', 'tested.', 'Our', 'results', 'suggest', 'that', 'a', 'complex', 'containing', 'TAF,', '110,', '63,', '48,', 'and', 'TBP', 'is', 'both', 'necessary', 'and', 'sufficient', 'to', 'provide', 'SLI', 'function.', 'Using', 'a', 'combination', 'of', 'epitope-tagged', 'versions', 'of', 'the', 'recombinant', 'TAF)s,', 'we', 'have', 'shown', 'that', 'each', 'of', 'the', 'subunits', 'of', 'SLi', 'can', 'individually', 'bind', 'to', 'TBP.', 'In', 'addi-', 'tion,', 'the', 'TAF\\s', 'appear', 'to', 'contact', 'each', 'other', 'in', 'order', 'co', 'form', 'a', 'relatively', 'stable', 'complex', 'that', 'presumably', 'involves', 'multiple', 'interactions.', 'These', 'TBP-TAF,', 'and', 'TAF,-', 'TAF,', 'interactions', 'do', 'not', 'require', 'DNA', '(5).', 'However,', 'it', 'was', 'of', 'critical', 'importance', 'to', 'determine', 'whether', 'these', 'protein-protein', 'contacts', 'resulted', 'in', 'a', 'functional', 'SL]', 'com-', 'plex', 'thar', 'could', 'support', 'transcription', 'of', 'the', 'ribosomal', 'promoter', 'by', 'RNA', 'pol', 'I.', 'Our', 'initial', 'attempts', 'to', 'assemble', 'functional', 'com-', 'plexes', 'from', 'TAF,s', 'expressed', 'in', 'Escherichia', 'coli', 'or', 'in', 'insect', 'cells', 'infected', 'with', 'recom-', 'binant', 'baculoviruses,', 'failed', 'to', 'produce', 'tran-', 'scriptionally', 'active', 'SL1.', 'One', 'reason', 'for', 'our', 'inability', 'to', 'reconstitute', 'active', 'SL1', 'com-', 'plexes', 'was', 'the', 'difficulty', 'in', 'obtaining', 'suffi-', 'ciencly', 'high', 'concentrations', 'of', 'the', 'four', 'sub-', 'units', 'of', 'SL1', 'in', 'order', 'to', 'perform', 'the', 'assem-', 'bly', 'reactions.', 'The', 'bacterially', 'expressed', 'pro-', 'teins', 'were', 'largely', 'insoluble,', 'and', 'the', 'amounts', 'of', 'soluble', 'baculovirus', 'expressed', 'TAF,s', 'were', 'limited.', 'Consequently,', 'we', 'tried', 'alternative', 'means', 'of', 'assembling', 'SLI', 'from', 'the', 'recombinant', 'subunits.', 'We', 'approached', 'the', 'problem', 'with', 'two', 'complementary', 'strat-', 'egies:', 'in', 'vivo', 'assembly', 'by', 'co-expression', 'of', 'the', 'subunits', 'and', 'in', 'vitro', 'assembly', 'of', 'dena-', 'tured', 'and', 'renatured', 'purified', 'subunits.', 'In', 'order', 'to', 'attempt', 'in', 'vivo', 'assembly', 'of', 'the', 'four', 'components', 'of', 'SL1,', 'we', 'generated', 'recombinant', 'vaccinia', 'viruses', 'that', 'would', 'di-', 'rect', 'the', 'production', 'of', 'each', 'subunit', 'in', 'HeLa', 'cells', 'upon', 'co-infection', 'with', 'the', 'various', 'combinations', 'of', 'viruses.', 'We', 'constructed', 'the', 'appropriate', 'vaccinia', 'virus', 'vectors', 'such', 'that', 'the', 'recombinant', 'TAF', 'could', 'be', 'distin-', 'guished', 'from', 'endogenous', 'TAF;s', 'on', 'the', 'basis', 'of', 'differences', 'in', 'molecular', 'weight', 'and', 'anti-', 'body', 'specificity', 'by', 'fusing', 'a', 'unique', 'epitope', 'tag', 'to', 'each', 'subunit.', 'TAF,48', 'was', 'constructed', 'with', 'a', 'FLAG-tag,', 'TAF,63', 'was', 'fused', 'to', 'the', 'Polyoma-Myc', '(PM)', 'epitope,', 'and', 'TAF,110', 'was', 'linked', 'to', 'the', 'hemagglutinin-peptide', '2015', ' ']