ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
J. Ciesiołka
SL18. The Role of Divalent Metal Ions in Functioning of
the Antigenomic Delta Ribozyme
Laboratory of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Noskowskiego 12/14, 61-704 Poznań,
e-mail: ciesiolk@ibch.poznan.pl
In the genomic RNA strand of the hepatitis delta virus (HDV), as well as in its antigenomic counterpart
generated during virus replication via the double rolling circle mechanism, there are two sequences with
ribozyme activities, called the delta ribozymes. Despite large progress in elucidation of the structure and
mechanism of catalysis of delta ribozymes, one of the most important issues, concerning the role of divalent
metal ions in their functioning, is still a mater of debate [1]. In our earlier studies we have compared the activity
of closely related variants of the antigenomic ribozyme in the presence of various divalent metal ions [2]. The
ribozymes differed in regions that were not directly involved in formation of the delta ribozyme catalytic core.
Thus the role of these peripheral elements in modulating ribozyme activity could be assessed. Interestingly, some
antibiotics and their complexes with metal ions could inhibit catalytic activity of this ribozyme [3].
The existing data on delta ribozymes do not show whether a similar or better ribozyme performance could be
achieved by catalytic centers that are composed of nucleotides other than the wild-type residues. High sequence
conservation of ribozyme regions of viral RNAs precludes answering this question. Simultaneous testing of a
very large number of ribozyme variants with multiple mutations is, however, possible with the use of the in vitro
selection methodology.
We used the in vitro selection method to search for catalytically active variants of the antigenomic delta
ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2 [4]. In the
initial combinatorial library sixteen nucleotide positions were randomized and the library contained a full
representation of all possible sequences. Following ten cycles of selection-amplification several catalytically
active ribozyme variants were identified. It turned out that one-third of the variants contained only single
mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next onethird
of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage
mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position of
the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’
and the capacity of functional adaptation [4]. In subsequent studies four selected ribozyme variants were
subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards
Mg 2+ , Ca 2+ and Mn 2+ ions. In order to localize tight metal ions binding sites within the ribozyme structures we
used the metal ion-induced cleavage method. Furthermore, in an attempt to analyze the importance of phosphate
oxygen atoms in both tertiary interactions and coordination of metal ions several NAIM (nucleotide analog
interference mapping) experiments were performed. The differences in catalytic activity of ribozyme variants
seem to be a consequence of the different abilities of various metal ions both to perform a chemical reaction as
well as to aid the formation of ribozyme structural core.
Acknowledgement: I would like to thank my present and former coworkers for their work with the delta
ribozymes and members of Prof. M. Jeżowska-Bojczuk group from University of Wrocław for collaboration.
This work was supported by the Polish Ministry of Science and Higher Education.
References:
[1] M.D. Been. CTMI 307, 47 (2006).
[2] J. Wrzesiński, M. Łęgiewicz, B. Smólska, J. Ciesiołka. Nucleic Acids Res. 29, 4482 (2001).
[3] J. Wrzesiński, M. Brzezowska, W. Szczepanik, M. Jeżowska-Bojczuk, J. Ciesiołka. Biochem. Biophy. Res.
Commun. 349, 1394 (2006).
[4] M. Łęgiewicz, A.Wichłacz, B. Brzezicha, J. Ciesiołka. Nucleic Acids Res. 34, 1270 (2006).
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