THE IMPORTANCE OF THE HELIX-2 REGION FOR THE CIS-CLEAVING AND TRANS-CLEAVING ACTIVITIES OF HEPATITIS-DELTA VIRUS RIBOZYMES

The sequence, secondary structure, and size requirements of the helix 2 region (H2) of a cis-acting hepatitis delta virus ribozyme Rz 1 were examined in this study. Mutational analysis was performed, and the cl eavage rate of each H2 mutant of Rz 1 was assayed. We found that H2 co uld be elongated to twice its original size without affecting ribozyme folding while the shortening of H2 by one base pair severely decrease d autolytic activity. In addition, the maintenance of the Watson-Crick base-pairing interactions of the last base pair of H2 (A16U58) was no t critical for cis-cleavage reaction. Nevertheless, mutants with an AA , an AG, an AC, or a GG pair at the bottom of H2 were less active, and the sequence of the H2/H3 interface might affect the stability of the catalytic core. The negative effects on ribozyme folding, such as the destabilization of H2, the unfavorable sequences at the last base pai r of H2 as well as the disruption of the continuity of H2 and H3, coul d be compensated for by elongating the H2 region of the corresponding mutants. The extension of H2 may alter the conformation of ribozyme mo lecules; in addition, it stabilized the catalytic core and enhanced th e resistance to formamide. Finally, for a transacting ribozyme and its substrate that require the formation of HI, H2, and H4 to reconstitut e the autocatalytic domain of HDV RNA, the extension of H2 stabilized the substrate/ribozyme complex and speeded up the cleavage rate but hi ndered the product release process.