RNA polymerase subunit RPB5 plays a role in transcriptional activation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 26; pp. 15281 - 15286
• Main Authors: Miyao, TG, Woychik, N.A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 22-12-1998; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Amino acids; Biochemistry; Biological Sciences; Cell growth; Chimeras; Conserved Sequence; DNA-directed RNA polymerase; enzyme activity; Fungal Proteins - metabolism; Genes; Genetic mutation; Genotype; Humans; Hybridity; initiation; Kinetics; Macromolecular Substances; messenger RNA; mutagenesis; Mutagenesis, Site-Directed; mutants; Plasmids; Polymerase Chain Reaction; promoter regions; Promoter Regions, Genetic; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; recombinant proteins; Ribonucleic acid; RNA; RNA Polymerase I - chemistry; RNA Polymerase I - metabolism; RNA Polymerase II - chemistry; RNA Polymerase II - metabolism; RNA Polymerase III - chemistry; RNA Polymerase III - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Social interaction; Trans-Activators - metabolism; transcription (genetics); Transcription, Genetic; Transcriptional Activation; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.26.15281

A mutation in RPBS (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPH1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.