MECHANISM OF RIBOSE 2'-GROUP DISCRIMINATION BY AN RNA-POLYMERASE

The mechanism by which T7 RNA polymerase (RNAP) discriminates between rNTP and dNTP substrates has been characterized. During transcript elo ngation T7 RNAP uses rNTPs 70-80-fold more efficiently than dNTPs. Dis crimination of the hydrogen-bonding character of the ribose 2'-substit uent contributes a largely K-m-mediated factor of similar to 20 to thi s preference for rNTPs, Discrimination of 2'-substituent H-bonding cha racter appears to be made through a hydrogen bond to the hydroxyl grou p of tyrosine 639. This hydrogen bond makes little net contribution to either rNTP ground or transition state binding energy apparently beca use it is balanced by the energy of desolvation of the tyrosine hydrox yl. This mechanism may reflect a strategy to facilitate translocation by minimizing contributions from polymerase-NMP moiety interactions to NTP binding energy so as to minimize the affinity of the NTP binding site for the 3'-NMP of the product nucleic acid.