Mutations in the Saccharomyces cerevisiae RPB1 gene conferring hypersensitivity to 6-azauracil.
RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.
Malagon
Francisco
F
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.
Kireeva
Maria L
ML
Shafer
Brenda K
BK
Lubkowska
Lucyna
L
Kashlev
Mikhail
M
Strathern
Jeffrey N
JN
D000595:Amino Acid Sequence; D000963:Antimetabolites / Q000494:pharmacology; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017354:Point Mutation; D012313:RNA / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology
D000963:Antimetabolites; D010455:Peptides; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae; C100248:azauracil