THE STRUCTURE OF THE L3 LOOP FROM THE HEPATITIS-DELTA VIRUS RIBOZYME - A SYN CYTIDINE

The structure of the L3 central hairpin loop isolated from the antigen omic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determine d by NMR spectroscopy, The 26 nt stem-loop structure contains nine bas e pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme, The structure was det ermined using homonuclear and heteronuclear NMR techniques on non-labe led and N-15-labeled RNA oligonucleotides. The overall root mean squar e deviation for the structure was 1.15 Angstrom (+/- 0.28 Angstrom) fo r the loop and the closing C.G base pair and 0.90 A (+/- 0.18 Angstrom ) for the loop and the closing C.G base pair but without the lone puri ne in the loop, which is not well defined in the structure. The struct ure indicates a U.C base pair between the nucleotides on the 5'- and 3 '-ends of the loop. This base pair is formed with a single hydrogen bo nd involving the cytosine exocyclic amino proton and the carbonyl O4 o f the uracil. The most unexpected finding in the loop is a syn cytidin e. While not unprecedented, syn pyrimidines are highly unusual. This o ne can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar stu dy of the structure of this loop showed a somewhat different three-dim ensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.