期刊
NUCLEIC ACIDS RESEARCH
卷 30, 期 12, 页码 2678-2685出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkf371
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We have determined the structures of complexes between the phage MS2 coat protein and variants of the replicase translational operator In order to explore the sequence specificity of the RNA-protein Interaction. The 19-nt RNA hairpins studied have substitutions at two positions that have been shown to be important for specific binding. At one of these positions, -10, which Is a bulged adenosine (A) In the stem of the wild-type operator hairpin, substitutions were made with guanosine (G), cytidine (C) and two non-native bases, 2-aminopurine (2AP) and Inosine (1). At the other position, -7 in the hairpin loop, the native adenine was substituted with a cytidine. Of these, only the G-10, C-10 and C-7 variants showed interpretable density for the RNA hairpin. In spite of large differences in binding affinities, the structures of the variant complexes are very similar to the wild-type operator complex. For G-10 substitutions in hairpin variants that can form bulges at alternative places in the stem, the binding affinity is low and a partly disordered conformation is seen In the electron density maps. The affinity Is similar to that of wild-type when the base pairs adjacent to the bulged nucleotide are selected to avoid alternative conformations. Both purines bind in a very similar way in a packet in the protein. In the C-10 variant, which has very low affinity, the cytidine Is partly inserted In the protein pocket rather than intercalated In the RNA stem. Substitution of the wild-type adenosine at position -7 by pyrimidines gives strongly reduced affinities, but the structure of the C-7 complex shows that the base occupies the same position as the A-7 In the wild-type RNA. It is stacked in the RNA and makes no direct contact with the protein.
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