DIMERIZATION OF MOMULV GENOMIC RNA - REDEFINITION OF THE ROLE OF THE PALINDROMIC STEM-LOOP H1-(278-303) AND NEW ROLES FOR STEM-LOOPS H2-(310-352) AND H3-(355-374)

Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structur e (H1) located in the encapsidation domain Psi (nucleotides 215-564) w as postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RN A dimerization, we systematically investigated the dimerization capaci ty of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by me asuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the spec ificity of dimerization of RNA nucleotides 1-725 is driven by motifs H 1-H3 in domain Psi. To define the-relative contributions of these elem ents, RNA deletion mutants containing different combinations of H1-H3 were-constructed and further analyzed in competition and kinetic exper iments; Our results confirm the importance of H1 in triggering dimeriz ation and shed new light on the mechanism of dimerization. H1 is requi red to provide a stable dimer, probably through the formation of exten ded intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser exte nt by H2. We suggest that they facilitate the recognition between the two RNAs, most Likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely rel ated processes.