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Olga Fedorova Research
Scientist
olga.fedorova@yale.edu
Domain
3: a catalytic effector in the group II ribozyme active site
Group II introns are self-splicing RNAs that are found in bacteria and
in the organellar genes of plants, fungi, yeast and algae. One of the
most intriguing features of group II introns is the similarity of their
self-splicing mechanism to that of pre-mRNA splicing, which suggests that
the spliceosomal machinery and group II introns might have evolved from
a common ancestor. Some group II introns are also mobile genetic elements
that insert themselves into double-stranded DNA as a unique type of retroelement.
Despite diversity in primary sequence, the secondary structure of group
II intron RNAs is very conserved. It is typically described as a series
of six domains that project from a central wheel. Most structural and
mechanistic studies of the intron have focused on domains 1 and 5, which
contain the nucleotide residues essential for catalysis, and on domain
6, which contains the branch-point adenosine. The functions of domains
2 and 3 (D2 and D3) are not yet well established. D3 can be considered
a "catalytic effector" because its presence in group II ribozyme constructs
dramatically increases the chemical reaction rate. However, the mechanism
by which it helps to accelerate group II catalysis remains unknown, and
there are no defined interactions between D3 and the rest of the intron.
D3 stimulates the chemical rate constant of group II intron reactions,
therefore behaving as a catalytic effector. However, D3 is unable to associate
independently with the ribozyme core. Recently, we have shown that docking
of D3 is mediated by a short duplex that is found at the base of D2. Nucleotide
analog interference mapping suggests an interaction between the D2 stem
and D3 that builds on the known ' interaction and extends it into D3 (1).
In addition to recruiting D3 into the core, the D2 stem contributes to
5'-splice site docking and ribozyme conformational change. Since D3 appears
to be one of the critical elements of the group II intron active site,
it is also important to elucidate how it interacts with other intronic
domains and which interactions mediate its function as a catalytic effector.
Phylogenetic data suggest that D3 does not bind to other domains via Watson-Crick
base-pairing. Therefore, in order to interact with the rest of the intron,
it must form a network of unusual long-range tertiary contacts. NAIM analysis
has established three catalytically important regions in D3: the internal
bulge, the pentaloop capping stem A and the single-stranded region between
the basal stem and stem A. By analyzing the interference pattern, we were
able to suggest the most likely conformation for the internal bulge and
the pentaloop of D3. In order to identify the interaction partners for
these areas, catalytically important nucleotides have been mutated, and
the mutants were subjected to NAIS assay. As a result, we have found the
first direct long-range tertiary contact between the catalytic domain
D5 and the catalytic effector D3. At the same time, in collaboration with
Dr. Steitz's lab, we are trying to obtain direct structural information
about the intron catalytic core. Although we have encountered a series
of difficulties in our attempts to crystallize the ribozyme construct
derived from the ai5 group II intron, we were able to obtain new information
on how the ribozyme folds. In particular, we have found that in the process
of folding the ribozyme forms a transient intermediate, the "near-native"
state, which we were able to trap and characterize its thermodynamic stability.
1. Fedorova, O., Mitros, T., and Pyle, A.M. (2003). Domains 2 and 3 interact
to form critical elements of the group II intron active site. J. Mol.
Biol., 330(2):197-209 |
