A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.
A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.
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在锤头状核酶裂解反应中，pH 依赖性构象变化（而不是化学步骤）似乎是限速因素。
DOI：
10.1006/jmbi.2001.5145
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Scott,WilliamG
中科院分区：
文献类型：
--
作者：
Murray,JamesB;Dunham,ChristineM;Scott,WilliamG
We have investigated the chemical basis for a previously observed 7.8 Å conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co2+) and requires the presence of an ionizable 2′-OH at the cleavage site, and note that this observed apparent pKaof 8.5 for the conformational change is within experimental error (±0.5) of the previously reported apparent kinetic pKaof 8.5 for the hammerhead ribozyme in the presence of Co2+. We have solved two crystal structures of hammerhead ribozymes having 2′-OCH3or 2′-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2′-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2′-oxygen drives this conformational change.
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DOI：
10.1107/s0907444995011115
发表时间：
1996-01-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Parkinson, G;Vojtechovsky, J;Berman, HM
通讯作者：
Berman, HM
影响因子：
4.5
作者：
O'Rear, JL;Wang, SL;Herschlag, D
通讯作者：
Herschlag, D
影响因子：
4.5
作者：
Curtis, EA;Bartel, DP
通讯作者：
Bartel, DP
DOI：
10.1146/annurev.biophys.27.1.475
发表时间：
1998
期刊：
Annual review of biophysics and biomolecular structure
影响因子：
--
作者：
J. Wedekind;David B. McKay
通讯作者：
J. Wedekind;David B. McKay
常规和时间分辨核酶 X 射线晶体学。
影响因子：
--
作者：
Scott,WG;Murray,JB
通讯作者：
Murray,JB