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Mutagenesis of a stacking contact in the MS2 coat protein‐RNA complex.
Author(s) -
LeCuyer K. A.,
Behlen L. S.,
Uhlenbeck O. C.
Publication year - 1996
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1996.tb01076.x
Subject(s) - biology , mutagenesis , stacking , rna , coat protein , coat , genetics , computational biology , mutation , gene , ecology , nuclear magnetic resonance , physics
The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single‐stranded pyrimidine in its RNA binding site has been examined. Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1–3 kcal/mol under standard binding conditions. Since the Phe, His and Cys 85 proteins formed UV photocrosslinks with iodouracil‐containing RNA at the same rate as the wild‐type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild‐type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.