Document 0168
 DOCN  M9590168
 TI    An improved version of the hairpin ribozyme functions as a
       ribonucleoprotein complex.
 DT    9509
 AU    Sargueil B; Pecchia DB; Burke JM; Department of Microbiology and
       Molecular Genetics, University of; Vermont, Burlington 05405, USA.
 SO    Biochemistry. 1995 Jun 13;34(23):7739-48. Unique Identifier : AIDSLINE
       MED/95298784
 AB    Most RNA molecules that are endowed with catalytic activity function in
       the form of ribonucleoproteins within cells. These complexes are
       frequently large, poorly defined, and difficult to study. As a model
       system to study biological catalysis by ribonucleoproteins, we have
       modified the hairpin ribozyme by inserting an RNA structure that serves
       as a binding site for bacteriophage R17 coat protein in the form of an
       extension to ribozyme helix 4, which lies at the periphery of the
       catalytic domain. In the absence of protein, we find that incorporation
       of the protein-binding domain increases the catalytic efficiency of the
       hairpin ribozyme by 2-fold for the cleavage reaction and 16-fold for the
       ligation reaction. This increase in activity correlates with an increase
       in the proportion of molecules which fold into the active tertiary
       structure, as measured by a UV cross-linking assay. Mobility-shift and
       filter-binding assays of complex formation show that R17 coat protein
       binds to the chimeric ribozyme with a dissociation constant essentially
       identical to that of the isolated protein-binding domain; no binding of
       the protein to the unmodified ribozyme could be detected. The kinetics
       of cleavage and ligation reactions are not altered by the presence of
       saturating concentrations of coat protein, and competition studies
       demonstrate that the protein remains bound to the ribozyme throughout
       the catalytic cycle. These studies establish that the hairpin ribozyme
       can be engineered to function efficiently in the form of a
       ribonucleoprotein in vitro and will serve as the basis for future
       experimentation to understand mechanisms of protein modulation of
       catalytic RNA activity, and to introduce other protein-binding domains,
       for example, HIV-1 rev-binding and tar elements, which may be useful for
       influencing subcellular localization, regulating intracellular activity,
       or generating ribozymes that also function as decoys in antiviral
       applications.
 DE    Base Sequence  Capsid/CHEMISTRY  Catalysis  Ligands  Molecular Sequence
       Data  Ribonucleoproteins/*CHEMISTRY  RNA-Binding Proteins/*CHEMISTRY
       RNA, Catalytic/*CHEMISTRY  Structure-Activity Relationship  Support,
       Non-U.S. Gov't  Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).