IDENTIFICATION OF MOLECULAR CONTACTS BETWEEN THE U1-A SMALL NUCLEAR RIBONUCLEOPROTEIN AND U1 RNA

被引:175
作者
JESSEN, TH
OUBRIDGE, C
TEO, CH
PRITCHARD, C
NAGAI, K
机构
[1] MRC Lab. of Molecular Biology, Cambridge CB2 2QH, Hills Road
关键词
MUTAGENESIS; RNA BINDING PROTEIN; STEM LOOP STRUCTURE; U1-A PROTEIN; U1; RNA; SNRNP;
D O I
10.1002/j.1460-2075.1991.tb04909.x
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
We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta-strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11 --> Val and Asn15 --> Val mutations completely abolished, and the Tyr13 --> Phe and Asn16 --> Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52 --> Gln abolished, but Arg52 --> Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop H and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A --> G and G --> A replacements in loop II, but not by C --> U or U --> C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.
引用
收藏
页码:3447 / 3456
页数:10
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