Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. Analysis with tetraloop mutants.
Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.
Glück
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A
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Endo
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Y
Wool
I G
IG
D001483:Base Sequence; D004722:Endoribonucleases; D004926:Escherichia coli; D005656:Fungal Proteins / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism / Q000648:ultrastructure; D012336:RNA, Ribosomal, 16S / Q000378:metabolism* / Q000648:ultrastructure; D012339:RNA, Ribosomal, 28S / Q000378:metabolism* / Q000648:ultrastructure; D012276:Ricin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005656:Fungal Proteins; D009841:Oligonucleotides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012339:RNA, Ribosomal, 28S; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases