C O M M U N I C A T I O N S
Figure 3. Active site of the Diels-Alderase ribozyme from the crystal
structure of the ribozyme-product complex5 with a proposed conformer of
the photoreactive product which is shown in the activated keteneimine form,
calculated using MOPAC: front view (left) and a 90° turned top view (right).
The two nucleotides U17 and C10 identified here are known to
be constituents of the active site and to make direct contacts with
the maleimide portion of the Diels-Alder product (Figure 3).5
In conclusion, we located the active site of the Diels-Alderase
ribozyme in solution by photoaffinity cross-linking. We identified
two specific nucleotides, C10 and U17, that are cross-linked by an
azide-substituted product analogue, and which of these two nucle-
otides is predominantly cross-linked depends on the Mg2+ ion
concentration. This metal-ion dependence reveals conformation-
dependent interactions between ribozyme and probe. While the
identified cross-link positions are in excellent agreement with the
available X-ray crystal structure, the design of photoaffinity probes
does not require such structural information. The results suggest
that for other ribozymes, a similar cross-linking approach could be
very informative even without a crystal structure available, which
is the situation for most other artificial ribozymes. The present work
demonstrates the utility of photoaffinity cross-linking as an empirical
approach that is applied here for the first time to an artificial
ribozyme. The ribozyme itself proved to be a useful catalyst for
the synthesis of the photoaffinity probe.
Figure 2. (a) Cross-linking of the bipartite ribozyme. Denaturing PAGE
autoradiogram of irradiated (indicated by flash) and nonirradiated samples
containing 5′-32P-labeled 38mer and different unlabeled 11-mer samples.
(b) Mg2+ ion dependence of cross-linking. Bands corresponding to cross-
linked species are indicated as XlnA, XlnB, and XlnC. (c) Mapping the
photoaffinity cross-link XlnA by limited alkaline hydrolysis.
(XlnC), and a rather diffuse band (XlnB) that disappears completely
with increasing Mg2+ concentration. The lower band (XlnC) shows
an intensity maximum at 8 mM Mg2+. The intensity of the upper
band (XlnA) increases continuously with the Mg2+ concentration
with a maximal cross-link yield at 80 mM where catalytic activity
is highest.
To determine the number of different species and the cross-link
position(s), the respective bands were excised from the gel, eluted,
32P-labeled at different positions, subjected to limited alkaline
hydrolysis, and reanalyzed by PAGE. Band XlnA derived from the
5′-32P-radiolabeled 38-mer and unlabeled 11-mer-DAN3 displayed
a large gap in the hydrolysis ladder starting at nucleotide U17
(Figure 2c). While for all nucleotides from nucleotide A16 down
to the 5′-end the hydrolysis bands comigrated with those of the
unmodified 38-mer, all bands from nucleotide U17 to the 3′-end
were retarded, strongly suggesting the attachment of the photoaf-
finity probe to position U17 in the 38-mer strand. At the cross-link
site, a tribranched structure is formed, resulting in RNA species
with lower electrophoretic mobility. Quantitative analysis of the
band densities by phosphorimaging indicates that U17 is the only
significant cross-link position inside this population. The cross-
link between the azidobenzyl group and U17 was further substanti-
ated by variations of the procedure, using either 3′-labeled 38-mer
or 3′-labeled 11-mer-DAN3. Applying the same methodology, the
lower cross-link band XlnC could be assigned to nucleotide C10,
whereas such an assignment failed for the diffuse band XlnB (Figure
2b).
Thus, using photoaffinity probe 11-mer-DAN3, three different
cross-links could be identified in this study, depending on the
conditions used: In the absence of divalent metal ions, cross-linking
is apparently unspecific. At intermediate metal ion concentrations,
nucleotide C10 is the predominant cross-linking site, while at high
concentration nucleotide U17 was preferentially cross-linked. This
metal-ion dependence indicates the existence of two different RNA
folds with affinity to the Diels-Alder product. Remarkably, the
metal ion dependent intensity changes of cross-link bands A, B,
and C parallel the changes in the proportions of folded, unfolded,
and intermediate populations, respectively, observed by single-
molecule FRET.4f This may suggest that the partially folded
intermediate FRET conformation is able to bind the Diels-Alder
product, which would explain why the ribozyme issdespite frequent
breakdown of the folded state4fsstrongly product-inhibited.4b
Acknowledgment. This work was supported by the Deutsche
Forschungsgemeinschaft, Human Fronties Science Program, and
the Fonds der Chemischen Industrie.
Supporting Information Available: Full experimental procedures
and analytical characterization. This material is available free of charge
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