Identification of global suppressors for temperature-sensitive folding mutations of the P22 tailspike protein.
Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84 greater than A, V331 greater than A, and A334 greater than V. The V331 greater than A or A334 greater than V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331 greater than A and A334 greater than V mutations were crossed into well-characterized temperature-sensitive folding (tsf) mutants at various sites in the tailspike protein, they suppressed all of the eight tsf mutants tested. Since the tsf defects destabilize folding intermediates rather than the native conformation, this result implies that the suppressors act in the folding pathway. Strains carrying the isolated suppressor mutations displayed no obvious phenotypic defect and formed native biologically active tailspikes. Thus, these single amino acid substitutions have striking influences on the efficiency of intracellular chain folding, without causing functional defects in the native protein.
Fane
B
B
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Villafane
R
R
Mitraki
A
A
King
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009154:Mutation; D012486:Salmonella typhimurium / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics*; D017376:Viral Tail Proteins
D000596:Amino Acids; D004269:DNA, Bacterial; D004279:DNA, Viral; D014764:Viral Proteins; D017376:Viral Tail Proteins