Using Phosphorothioate-Substituted RNA To Investigate the Thermodynamic Role of Phosphates in a Sequence Specific RNA−Protein Complex
- 9 December 1999
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 39 (1) , 55-63
- https://doi.org/10.1021/bi991769v
Abstract
Part of the binding affinity and specificity in RNA-protein complexes is often contributed by contacts between the protein and backbone phosphates that are held in position by the RNA structure. This study focuses on the well-characterized interaction between a dimer of the MS2 coat protein and a small RNA hairpin. Using a short oligoribonucleotide which contains all the necessary sequence elements required for tight protein binding, a single phosphorothioate linkage was introduced at 13 different positions. In each case, the R(P) and S(P) stereoisomers were separated and their affinities to the MS2 coat protein were determined. Comparison of these biochemical data with the crystal structure of the protein-hairpin complex indicates that introduction of a phosphorothioate only affects binding at sites where a protein-phosphate contact is observed in the crystal structure. This means that phosphorothioate-containing oligoribonucleotides should also be useful for mapping phosphate contacts in RNA-protein complexes for which no crystal structure is available.Keywords
This publication has 14 references indexed in Scilit:
- Structural effect of complete [Rp]-phosphorothioate and phosphorodithioate substitutions in the DNA strand of a model antisense inhibitor-target RNA complexJournal of Molecular Biology, 1999
- Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: Implications for binding requirementsRNA, 1999
- Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioatesJournal of Molecular Biology, 1998
- The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactionsJournal of Molecular Biology, 1997
- Synthesis of phosphorodithioate RNA by the H-phosphonothioate methodTetrahedron Letters, 1996
- Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction EndonucleasePublished by Elsevier ,1996
- Probing the Indirect Readout of the Restriction Enzyme EcoRVPublished by Elsevier ,1996
- The automated chemical synthesis of long oligoribuncleotides using 2'-O-silylated ribonucleoside 3'-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3'-half molecule of an Escherichia coli formylmethionine tRNAJournal of the American Chemical Society, 1987
- Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozymeNature, 1987
- Stereochemistry of a substrate for pancreatic ribonuclease. Crystal and molecular structure of the triethylammonium salt of uridine 2', 3'-0,0-cyclophosphorothioateJournal of the American Chemical Society, 1970