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PMID: 9003760 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.

The EMBO journal ·Vol. 15 ·No. 24 ·1996-12-16 ·Pages 6847-53

LeCuyer KA, Behlen LS, Uhlenbeck OC

Abstract

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.

MeSH Terms
Capsid/chemistry Capsid Proteins Cloning, Molecular Hydrogen-Ion Concentration Molecular Structure Mutagenesis Photochemistry RNA/chemistry RNA Probes RNA-Binding Proteins/chemistry Thermodynamics
Chemicals
Capsid Proteins RNA Probes RNA-Binding Proteins RNA
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
LeCuyer K A
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Behlen L S
Uhlenbeck O C
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-12-16
Pages
6847-53
Language
English
Region
England
NLM ID
8208664
PMCID
PMC452510
Subset
IM
Grants
NIGMS NIH HHS · GM36944 · United States
External Links
PubMed source PMC Full Text View journal details
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