HYDROGEN-BONDING CONTACTS IN THE MAJOR GROOVE ARE REQUIRED FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PROTEIN RECOGNITION OF TAR RNA

被引:117
作者
HAMY, F [1 ]
ASSELINE, U [1 ]
GRASBY, J [1 ]
IWAI, S [1 ]
PRITCHARD, C [1 ]
SLIM, G [1 ]
BUTLER, PJG [1 ]
KARN, J [1 ]
GAIT, MJ [1 ]
机构
[1] MRC,MOLEC BIOL LAB,HILLS RD,CAMBRIDGE CB2 2QH,ENGLAND
关键词
HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 (HIV-1); TRANSACTIVATION; RNA-PROTEIN INTERACTIONS; TAT PROTEIN; TAR RNA STRUCTURE;
D O I
10.1006/jmbi.1993.1129
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
The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotidesto form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR HNA. Substitutionat positions U23 or U25 bythe base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10− to 20−fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A singlemethylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful totat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23. © 1993 Academic Press, Inc.
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页码:111 / 123
页数:13
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