Flexible regions of RNA structure facilitate co-operative Rev assembly on the Rev-response element

The oligomerisation of Rev on the Rev-response element (RRE) was studied using a series of model substrates. Only a monomer of Rev is able to bind efficiently to a high affinity site that is flanked by perfect duplex RNA. Addition of a bulge or a second stem structure adjacent to the high affinity site permits the co-operative incorporation of a second Rev molecule to the RNA. Model RREs carrying bulges can bind Rev with a higher degree of co-operativity than the native structure. Oligomerisation was efficient when the bulge was moved to the opposite strand of the duplex, but was severely impaired when the distance between the bulge and the high affinity site was increased by more than 8 bp. Rev can oligomerise at either end of the RNA-protein complex formed at the high affinity site; when the duplex flanking a high affinity site is disrupted by a bulge or a stem, oligomerisation proceeds in the direction of the disruption regardless of the orientation of the high affinity site. The results are consistent with the "molecular rheostat" model for RRE function, which suggests that Rev binding to the RRE is highly distributive and provides a sensitive measurement of intracellular Rev concentrations.