S. Ananvoranich et al., Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem, NUCL ACID R, 27(6), 1999, pp. 1473-1479
Our previous report on delta ribozyme cleavage using a trans-acting antigen
omic delta ribozyme and a collection of short substrates showed that the mi
ddle nucleotides of the pi stem, the substrate binding site, are essential
for the cleavage activity. Here we have further investigated the effect of
alterations in the P1 stem on the kinetic and thermodynamic parameters of d
elta ribozyme cleavage using various ribozyme variants carrying single base
mutations at putative positions reported. The kinetic and thermodynamic va
lues obtained in mutational studies of the two middle nucleotides of the P1
stem suggest that the binding and active sites of the delta ribozyme are u
niquely formed. Firstly the substrate and the ribozyme are engaged in the f
ormation of a helix, known as the P1 stem, which may contain a weak hydroge
n bond(s) or a bulge. Secondly, a tertiary interaction involving the base m
oieties in the middle of the P1 stem likely plays a role in defining the ch
emical environment. As a consequence, the active site might form simultaneo
usly or subsequently to the binding site during later steps of the pathway.