A single amino acid substitution in ribonucleolytic toxin restrictocin abolishes its specific substrate recognition activity.
Restrictocin is a small basic protein produced by the fungus Aspergillus restrictus. It potently inhibits protein synthesis in eukaryotic cells by specifically cleaving a single phosphodiester bond in 28S rRNA. A histidine residue at position 49 in restrictocin has been implicated in its active site. A mutant of restrictocin in which the histidine at position 49 was changed to an alanine was constructed by site-directed mutagenesis, and the protein was expressed in Escherichia coli. The mutant and the wild type proteins were found to be structurally identical. Unlike restrictocin, the restrictocin H49A mutant did not cleave the ribosomal RNA specifically at the target phosphodiester bond; instead, it extensively degraded the RNA substrate with altered specificity. The mutant exhibited a high ribonuclease activity compared to restrictocin on yeast tRNA, and poly(U) and poly(C). The mutant also poorly inhibited protein synthesis in eukaryotic cells as well as in a cell free system. We therefore propose that histidine 49 of restrictocin is not involved per se in the enzymatic activity; however, it does play a crucial role in the specific recognition of the target sequence by restrictocin.
Nayak
S K
SK
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Batra
J K
JK
D000409:Alanine / Q000235:genetics; D000485:Allergens; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D052179:Antigens, Plant; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006367:HeLa Cells; D006639:Histidine / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism / Q000633:toxicity; D012331:RNA, Fungal / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011817:Rabbits; D012260:Ribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D005656:Fungal Proteins; D009183:Mycotoxins; D011500:Protein Synthesis Inhibitors; D012331:RNA, Fungal; D012339:RNA, Ribosomal, 28S; C036196:MITF protein, Aspergillus restrictus; D006639:Histidine; D012260:Ribonucleases; D000409:Alanine