RPAP1, a Novel Human RNA Polymerase II-Associated Protein Affinity Purified with Recombinant Wild-Type and Mutated Polymerase Subunits
AUTOR(ES)
Jeronimo, Célia
FONTE
American Society for Microbiology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=479746Documentos Relacionados
- Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity
- RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.
- Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.
- Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.
- Wild-type and mutant in vitro products of an operon for ribonucleic acid polymerase subunits.