Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.
Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.
Laboratorium für Biochemie, Universität Bayreuth, FRG.
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Förster
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Ott
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Forchhammer
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Sprinzl
D002846:Chromatography, Affinity; D003062:Codon; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*
D003062:Codon; D012345:RNA, Transfer, Amino Acid-Specific; C036928:tRNA, selenocysteine-; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu