METAL-ION REQUIREMENTS FOR SEQUENCE-SPECIFIC ENDORIBONUCLEASE ACTIVITY OF THE TETRAHYMENA RIBOZYME
METAL-ION REQUIREMENTS FOR SEQUENCE-SPECIFIC ENDORIBONUCLEASE ACTIVITY OF THE TETRAHYMENA RIBOZYME
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DOI：
10.1021/bi00443a017
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发表时间：
1989-08-22
期刊：
影响因子：
2.9
通讯作者：
CECH, TR
中科院分区：
文献类型：
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作者：
GROSSHANS, CA;CECH, TR
嗜热四膜虫的自剪接间插序列RNA的缩短形式充当酶，催化RNA底物的序列特异性切割。我们现在已经考察了这个反应对金属铁的要求。Mg 2+和Mn 2+是仅有的自身赋予RNA酶活性的金属离子。原子吸收光谱法表明，Zn，Cu，Co和Fe不是以与RNA酶等摩尔的量存在的，并且当添加到反应混合物中时不促进切割。因此，这些离子可以作为反应的辅因子被消除。虽然Ca2+本身没有活性，它的Mg2+需求的一部分，1 mM的Ca2+降低Mg2+的最佳从2到1 mM。这些结果，结合研究的金属离子的混合物的反应性，使我们假设，两类金属离子结合位点是催化所需的。1类位点与Mn 2+的活性大于与Mg 2+的活性，而其它二价离子以及Na+和K+没有活性。目前尚不清楚位于1类位点的离子是否具有特定的结构作用或直接参与活性位点化学。第2类位点，推测是结构性的，具有Mg 2 + ≥的优先顺序。Ca 2 + > Mn 2+和Ca 2 + > Sr 2 + > Ba 2+，Zn 2+、Cu 2+、Co 2+、Na+和K+在测试的浓度范围内没有给出可检测的活性。
A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal iron requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, led us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ .gtoreq. Ca2+ > Mn2+ and Ca2+ > Sr2+ > Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.