Bevilacqua P C, Johnson K A, Turner D H
Department of Chemistry, University of Rochester, NY 14627.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8357-61. doi: 10.1073/pnas.90.18.8357.
The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15 degrees C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate. Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.
通过在15摄氏度下进行荧光滴定和动力学实验，测定了5'-磷酸鸟苷和2'-脱氧-5'-磷酸鸟苷对芘标记的5'外显子模拟物（pyCUCU）与嗜热四膜虫催化RNA（核酶）结合的热力学和动力学的影响。在饱和5'-磷酸鸟苷存在下，pyCUCU与L-21 Sca I截短核酶的结合减弱了5倍，而在饱和2'-脱氧-5'-磷酸鸟苷存在下，其结合增强了4倍。动力学实验结果表明，5'-磷酸鸟苷存在下的反协同效应主要源于核酶催化核心与由pyCUCU和核酶的GGAGGG形成的P1双链体之间三级接触形成较慢。相反，2'-脱氧-5'-磷酸鸟苷存在下的协同效应主要源于核酶催化核心与P1双链体之间三级接触的破坏较慢。额外的实验表明，这些协同和反协同效应不是芘标记的作用，不是由盐效应引起的，也不是特定于核酶的一种复性程序。
