CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .2. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE THAT FORMS A MISMATCH AT THE ACTIVE-SITE
CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .2. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE THAT FORMS A MISMATCH AT THE ACTIVE-SITE
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DOI：
10.1021/bi00496a004
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发表时间：
1990-11-06
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：
HERSCHLAG, D;CECH, TR
由来自四膜虫pre-rRNA间插序列的核酶催化的位点特异性核酸内切酶反应的特征在于具有与核酶的5“外显子结合位点形成“匹配”双链体的底物[G2 CCCUCUA 5 + G.dblarw. G2 CCCUCU + GA 5（G =鸟苷）; Herschlag，D.，和Cech，T. R.（1990）生物化学（在此问题上的前一篇论文）]。饱和底物的限速步骤是产物G2 CCCUCU的解离。在此，我们表明，底物G2 CCCGCUA 5的反应具有约为λ的kcat值，所述底物G2 CCCGCUA 5与切割位点-3位的5 ″外显子结合位点形成“错配”双链体。102-比匹配底物（50 ℃，10 mM MgCl 2，pH 7）的kcat大一倍。这可以通过错配产物G2 CCCGCU比匹配产物更快解离来解释。在寡核苷酸底物亚饱和和G饱和的情况下，寡核苷酸底物的结合和化学步骤各自是部分限速的。内切核酸酶反应的化学步骤的速率常数和位点特异性水解反应的速率常数（其中溶剂代替G）各自在λ内。2-折叠与匹配和不匹配的基板，尽管。103-倍弱结合的错配基板。这可以被描述为在基态和过渡态中碱基在位置-3处的“均匀结合”[Albery，W. J.，和Knowles，J.R.（1976）Biochemistry 15，5631-5640]。因此，匹配的底物不使用其额外的结合能来优先稳定过渡态。产物G2 CCCGCU和G2 CUCUCU与核酶的结合强度低于匹配产物G2 CCCUCU。然而，像匹配的产品一样，每个都绑定. apprx。104-fold（. apprx. 6 kcal/mol）比预测的更强。这表明在位置-3或-5处碱基配对的破坏不干扰负责增强的结合稳定性的核酶的三级相互作用。与亚饱和错配底物反应的KmG = 110 μ M的值小于Kd的解离常数（E. G）. simeq. 500 μ M，因为限速步骤从低G下的化学变化到高G下寡核苷酸底物的（部分）结合。在低G下的限速化学允许增强对错配底物的区分，而无需求助于先前已用于增强特异性的添加的变性剂。提出了5“外显子沿着的强结合与G的弱结合以确保自剪接期间的有效外显子连接。
The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5'' exon binding site of the ribozyme [G2CCCUCUA5 + G .dblarw. G2CCCUCU + GA5 (G = guanosine); Herschlag, D., and Cech, T. R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5'' exon binding site at position -3 from the cleavage site, has a value of kcat that is .apprx. 102-fold greater than kcat for the matched substrate (50.degree.C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within .apprx. 2-fold with the matched and mismatched substrates, despite the .apprx. 103-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W. J., and knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state. The products G2CCCGCU and G2CUCUCU bind the ribozyme less strongly than the matched product, G2CCCUCU. Nevertheless, like the matched product, each binds .apprx. 104-fold (.apprx. 6 kcal/mol) stronger than predicted for base pairing between oligonucleotides. This suggests that disruption of base pairing at position -3 or -5 does not interfere with the tertiary interactions of the ribozyme that are responsible for the enhanced binding stability. The value of KmG = 110 .mu.M for the reaction with subsaturating mismatched substrate is less than the dissociation constant of Kd (E .cntdot. G) .simeq. 500 .mu.M because of change in rate-limiting step from chemistry at low G to (partially) binding of the oligonucleotide substrate at high G. Rate-limiting chemistry at low G allows enhanced discrimination against the mismatched substrate, without recourse to added denaturants, which have been used previously to enhance specificity. The strong binding of the 5'' exon along with the weak binding of G is proposed to ensure efficient exon ligation during self-splicing.