Importance of specific adenosine N-3-nitrogens for efficient cleavage by a hammerhead ribozyme

被引:23
作者
Bevers, S [1 ]
Xiang, GB [1 ]
McLaughlin, LW [1 ]
机构
[1] BOSTON COLL,DEPT CHEM,CHESTNUT HILL,MA 02167
关键词
D O I
10.1021/bi952868l
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N-3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazaadenosine (c(3)A) base analogue for residues A(6), A(9), A(13), A(14), or A(15.1). Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by k(cat)K(M), for the c(3)A(6), c(3)A(9), and c(3)A(14) complexes were only marginally reduced (less than or equal to 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c(3)A(13) and c(3)A(15.1) complexes were reduced by 9-fold and 55-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower k(cat) values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N-3-nitrogen of the A(13) residue and particularly the A(15.1) residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of thee critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization.
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页码:6483 / 6490
页数:8
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