Kinetic self-cleavage models for permuted variants of the antigenomic HDV ribozyme

The kinetic characteristics have been studied for noncircularly permuted va riants of the human hepatitis delta virus antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Differen t ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic co nstant of the first reaction phase and the portion of the ribozyme molecule s that self-cleaved during the first phase. Partial restoration of the init ial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the da ta obtained, a scheme of the self-cleavage reaction has been proposed inclu ding: (i) activation of the ribozyme with energy of 40-50 kcal/mol and a ch aracteristic time of several deciminutes under optimal reaction conditions, (ii) fast and reversible reaction of the phosphodiester bond cleavage; (ii i) reaction leading to isomerization of the 3',5'-phosphodiester bond to th e 2',5' bond in the self-cleavage site with a characteristic activation tim e of tens of minutes; and (iv) practically irreversible conformational chan ge leading to fixation of the cleavage by immobilization of the 5'-terminal nucleotide of the product in the center of the formed structure and displa cement of the 3'-terminal nucleotide to the periphery. The latter process h as a characteristic time of tens of minutes and a low activation energy.