hide
Free keywords:
-
Abstract:
The kinetics of the interaction of GTP and GDP with SelB, the specific
translation factor for the incorporation of selenocysteine into
proteins, have been investigated using the stopped-flow method. Useful
signals were obtained using intrinsic (i.e. tryptophan) fluorescence,
the fluorescence of methylanthraniloyl derivatives of nucleotides, or
fluorescence resonance energy transfer from tryptophan to the
methylanthraniloyl group. The affinities of SelB for GTP (K-d = 0.74 mu
M) and GDP (K-d = 13.4 mu M) were considerably lower than those of other
translation factors. Of functional significance is the fact that the
rate constant for GDP release from its complex with SelB (15 s(-1)) is
many orders of magnitude larger than for elongation factor Tu,
explaining why a GDP/GTP exchange factor is not required for the action
of SelB. In contrast, the rate of release of GTP is 2 orders of
magnitude slower and not significantly faster than for elongation factor
Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that
represents a binding site for the protein and that is part of the fdhF
selenocysteine insertion sequence element positioned immediately
downstream of the UGA triplet coding for selenocysteine incorporation,
the kinetics of the interaction were studied. The high affinity of the
interaction (K-d similar to 1 nM) appeared to be increased even further
when seleno-cysteyl-tRNA(Sec) was bound to SelB, but to be independent
of the presence or nature of the guanosine nucleotide at the active
site. These results suggest that the affinity of SelB for its RNA
binding site is maximized when charged tRNA is bound and decreases to
allow dissociation and reading of codons downstream of the
selenocysteine codon after selenocysteine peptide bond formation.
