Glück A, Endo Y, Wool I G
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
J Mol Biol. 1992 Jul 20;226(2):411-24. doi: 10.1016/0022-2836(92)90956-k.
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.
蓖麻毒素是一种细胞毒性蛋白，它通过水解真核生物28S rRNA中第4324位腺苷的碱基与核糖之间的N-糖苷键来使核糖体失活。蓖麻毒素A链也会催化裸露的原核生物16S rRNA中的脱嘌呤作用；腺苷位于GAGA四环中的第1014位。使用合成的GAGA四环寡核糖核苷酸研究了蓖麻毒素A链识别和催化切割的rRNA识别元件。指定为野生型的RNA，一种近似16S rRNA中蓖麻毒素敏感位点结构的寡核糖核苷酸（19聚体），以及一些突变体，用噬菌体T7 RNA聚合酶从合成DNA模板体外转录。对于野生型四环寡核糖核苷酸，蓖麻毒素A链催化的反应的Km为5.7微摩尔，Kcat为0.01分钟-1。毒素α-肌动蛋白，它切割28S rRNA中G4325 3'侧的磷酸二酯键，不识别四环RNA，尽管α-肌动蛋白确实会影响一个更大的合成寡核糖核苷酸，该寡核糖核苷酸有一个带有GAGA序列的17个核苷酸的环；因此，这两种毒素的识别元件存在明显差异。构建了GAGA四环中每个核苷酸所有可能的转换和颠换的突变体；没有一个被蓖麻毒素A链识别。因此，四环中GAGA序列的完整性是绝对必要的。四环寡核糖核苷酸的螺旋茎可以减少到三个碱基对，如果温度降低，实际上可以减少到两个碱基对，而不影响识别；这些碱基对的性质不影响蓖麻毒素A链的识别或催化。如果四环打开形成一个含GAGA的六环，蓖麻毒素A链的识别就会丧失。这表明在延伸周期中，一个GAGA四环要么存在于28S rRNA蓖麻毒素结构域假定的17个成员的单链区域中，要么在其中形成，这与蛋白质合成机制有关。
