RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo.
RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Deltarpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Deltarpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Deltarpb9 cells revealed only a few changes, predominantly in genes related to metabolism.
Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G 1L6, Canada.
S A
SA
Hemming
D B
DB
Jansma
P F
PF
Macgregor
A
A
Goryachev
J D
JD
Friesen
A M
AM
Edwards
D000409:Alanine / Q000737:chemistry; D000483:Alleles; D000963:Antimetabolites / Q000494:pharmacology; D002455:Cell Division; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000378:metabolism; D017353:Gene Deletion; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry
D000963:Antimetabolites; D018076:DNA, Complementary; D005656:Fungal Proteins; D014498:Uracil; D012313:RNA; D012319:RNA Polymerase II; C100248:azauracil; D015032:Zinc; D000409:Alanine