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.

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.