lication. The kinetics of the interaction between RNAI and the nascent RNAH have been studied in great detail by Tomizawa who showed that the process occurs in two steps. The first step is faster and reversible and involves the hydrogen bonding of some nucleotides in 
the three complementary loops. The end product, however, is a complete hybrid between the two RNAs. The second inhibitor of plasmid replication has been identified as a protein of 63 amino acids. The gene encoding it has been named Rop since it was observed that it can negatively regulate the expression of genes under the control of the primer promoter. In vivo and in vitro studies however have pointed out that Rop does not act as a classical repressor PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19827996 but enhances the inhibitory activity of RNAI. Tomizawa and Som have shown that, in the presence of Rop, the hybridization of RNAI with the primer occurs faster than in its absence and, more specifically, that Rop increases the rate of the first reversible step of the interaction. Because of this observation these authors have changed the name of the protein into Rom to underline its better defined function. The hybridization mechanism regulated by Rop is not the only molecular mechanism that depends on the hybridization of two RNAs and is regulated by a protein. Recently the initiator protein of the ColEl-unrelated plasmid R6K and the NBP.P12 protein of the avian retrovirus have been shown to have RNA annealing activity. 621 L.Castagnoli et al. The structure of Rop. Stereo diagram of the Rop monomer. The monomer is viewed from the outside of the molecule, the 2-fold axis being horizontal behind the molecule. Residue numbers are indicated. We have recently shown that Rop specifically interacts with the stem structures of RNAI and RNAII and we have proposed that Rop performs its function by acting as an adaptor for the correct positioning of the two RNA molecules. Here we discuss the high resolution structure of Rop and we analyse the phenotype of several mutants in which CJ-023423 solvent exposed side chains have been altered. Ndel Fspl 3000 Results and Discussion The three dimensional structure of Rop To elucidate the function of Rop at the molecular level we have overexpressed, purified and solved by X-ray crystallography the three-dimensional structure of the protein. The native Rop is a dimer as shown by its migration properties in a gel filtration column and by its structure in crystals. Each of the two subunits folds in a very simple and regular structure constituted by two a-helices connected by a sharp bend. The two subunits assemble in the dimer to form a bundle of four a-helices with an exact 2-fold symmetry axis. The resulting molecule is very compact and stable to heat and chemical denaturants. The samples were treated in different conditions with iodo-acetic acid and electrophoresed overnight at 50 V on a 0.1 mm thick gel. Panel B the five samples were incubated with different concentration ratios of iodo-acetic acid and iodoacetamide. The latter chemical blocks the thiol groups without changing their charge. The figures on the left of the gel indicate the number of extra carboxyl groups corresponding to each band. Panel C, the denaturation of Rop is reversible in this system. The in vitro translation products were heated at three different temperatures and then cooled down to 37C. The seven samples, native control and denatured and renatured protein at the three different temperatures, were all treated for 10 min at 37C w