Viroids and other circular subviral
RNA pathogens, such as the
hepatitis delta agent, use a rolling circle replication cycle requiring an intact
circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta
RNA contains a highly conserved
element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta
ribozyme, demonstrating that the
element of local tertiary structure can function as a
ribozyme control
element in vitro. Physical studies of the complex containing this
element were carried out. The complex binds
magnesium ions and is not readily dissociated by
EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM
EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of
sodium ions.
A DNA complex and a perfect
RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The
RNA complex melts at temperatures approximately 10 degrees C lower in
buffers containing 0.5 mM
MgCl2 and 100 mM NaCl than in
buffers containing 0.5 mM
MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The
element of local tertiary structure in delta genomic
RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.