Abstract
We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Binding Sites
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Carrier Proteins / genetics
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Carrier Proteins / isolation & purification*
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Carrier Proteins / metabolism*
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DNA / metabolism
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Expressed Sequence Tags
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Gene Expression Regulation
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Histones / metabolism
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Humans
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Models, Molecular
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Molecular Sequence Data
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Multienzyme Complexes
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Mutation*
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Phosphoprotein Phosphatases / isolation & purification
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Phosphoprotein Phosphatases / metabolism
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Promoter Regions, Genetic
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Protein Conformation
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Protein Subunits / genetics
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Protein Subunits / isolation & purification*
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Protein Subunits / metabolism*
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RNA Polymerase II / genetics
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RNA Polymerase II / isolation & purification*
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RNA Polymerase II / metabolism*
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Sequence Homology, Nucleic Acid
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Transcription Factor TFIIB / genetics
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Transcription Factor TFIIB / isolation & purification
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Transcription Factor TFIIB / metabolism
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Transcription Factors, TFII / genetics
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Transcription Factors, TFII / isolation & purification
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Transcription Factors, TFII / metabolism
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Transcription, Genetic
Substances
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Carrier Proteins
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Histones
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Multienzyme Complexes
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Protein Subunits
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RPAP1 protein, human
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factor TFIIB
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Transcription Factors, TFII
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DNA
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RNA Polymerase II
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Phosphoprotein Phosphatases
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carboxy-terminal domain phosphatase
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transcription factor TFIIF