Determination of RNA–Protein Contacts Using Thiophosphate Substitutions

The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA–protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure that contains four essential single-stranded nucleotides. Additional specificity is thought to be due to four or five ionic contacts between the protein and phosphates on the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5′-O-(1-thiotriphosphates) [NTP(αS)s], allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site were synthesized with different NTP(αS)s and tested for coat protein binding to deduce positions of thiophosphates that alter the binding affinity. Of the twenty-one phosphate positions in the molecule, two were found to decrease the K_a3-fold when substituted with a thiophosphate, one position decreased the K_a10-fold, and one position increased the K_a10-fold. Substitution of any of the other 17 positions with thiophosphates does not alter the K_a. The four positions that alter the K_aare located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates affect binding because they contact coat protein directly.