FLEXIBLE REGIONS OF RNA STRUCTURE FACILITATE COOPERATIVE REV ASSEMBLYON THE REV-RESPONSE ELEMENT

The oligomerisation of Rev on the Rev-response element (RRE) was studi ed using a series of model substrates. Only a monomer of Rev is able t o 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 se cond Rev molecule to the RNA. Model RREs carrying bulges can bind Rev with a higher degree of co-operativity than the native structure. Olig omerisation was efficient when the bulge was moved to the opposite str and 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 a t the high affinity site; when the duplex flanking a high affinity sit e is disrupted by a bulge or a stem, oligomerisation proceeds in the d irection of the disruption regardless of the orientation of the high a ffinity 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 intrace llular Rev concentrations. (C) 1996 Academic Press Limited