Involvement of loops L2 and L4 of ribonucleolytic toxin restrictocin in its functional activity.
Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. The long loops in restrictocin molecule have been shown structurally to be involved in target RNA recognition. In this study we have investigated the role of some putative substrate-interacting residues in loops L2 and L4, spanning residues 36-48 and 99-117, respectively in restrictocin catalysis. The residues Lys42, Ser46, Pro48 and Lys111 were individually mutated to alanine to probe their role in restrictocin function. The mutation of Lys111 to alanine, although did not affect the ribonucleolytic activity, rendered the toxin completely inactive in inhibiting translation in HeLa cells as well in an in vitro cell free system. The loop L4 in restrictocin appears to be more critical compared to loop L2 for its interaction with the specific substrate.
Dey
Punyatirtha
P
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Tripathi
Manisha
M
Batra
Janendra K
JK
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009183:Mycotoxins / Q000737:chemistry / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005656:Fungal Proteins; D009183:Mycotoxins; D011500:Protein Synthesis Inhibitors; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; C036196:MITF protein, Aspergillus restrictus; D012694:Serine; D012313:RNA; D011392:Proline; D012260:Ribonucleases; D008239:Lysine