Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem

Citation
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
Citations number
15
Categorie Soggetti
Biochemistry & Biophysics
Journal title
NUCLEIC ACIDS RESEARCH
ISSN journal
03051048 → ACNP
Volume
27
Issue
6
Year of publication
1999
Pages
1473 - 1479
Database
ISI
SICI code
0305-1048(19990315)27:6<1473:MAOTAT>2.0.ZU;2-5
Abstract
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.