Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae.
The MAK3 gene of Saccharomyces cerevisiae is necessary for the propagation of the L-A double-stranded RNA virus and its satellites, such as M1 that encodes a killer toxin. We cloned the MAK3 gene based on its genetic map position using physically mapped lambda-clones covering nearly all of the yeast genome. The minimal sequence necessary to complement the mak3-1 mutation contained 3 open reading frames (ORFs). Only one (ORF3) was necessary to complement mak3-1. A deletion insertion mutant of ORF3 grew slowly on nonfermentable carbon sources, an effect not due simply to its loss of L-A. Although ORF3 alone is sufficient for MAK3 activity when expressed from an expression vector, in its native context an additional 669 base pairs 3' to the ORF and complementary to the gene for a non-histone protein are necessary for expression, but not for normal steady state transcript levels. This suggests a post-transcriptional control of MAK3 expression by the 3' region. The MAK3 protein has substantial homology with several N-acetyltransferases with consensus patterns h..h.h. . . Y..[HK]GI[AG][KR].Lh. . .h and h.h[DE]. . . .N..A. . .Y . . .GF. . . .. . . .Y . . [DE]G, (h = hydrophobic). Mutation of any of the underlined conserved residues (94GI----AA, 123N----A, 130Y----A, 134GF----SL, 144Y----A, and 149G----A) inactivated the gene, supporting the hypothesis that MAK3 encodes an N-acetyltransferase.
Tercero
J C
JC
Section on Genetics of Simple Eukaryotes, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Riles
L E
LE
Wickner
R B
RB
D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002874:Chromosome Mapping; D016386:Chromosome Walking; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D010957:Plasmids; D012328:RNA Viruses / Q000502:physiology*; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014779:Virus Replication
D004271:DNA, Fungal; D005656:Fungal Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C077299:MAK3 protein, S cerevisiae; D001191:Arylamine N-Acetyltransferase