Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.
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 micrometer) and GDP (K(d) = 13.4 micrometer) 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) approximately 1 nm) appeared to be increased even further when selenocysteyl-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.
Thanbichler
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Lehrstuhl für Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, 80638 München, Germany.
Bock
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Goody
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D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000378:metabolism
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; C061878:SelB protein, Bacteria; D062367:ortho-Aminobenzoates; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan