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Zusammenfassung:
The cotranslational incorporation of selenocysteine into proteins is
mediated by a specialized elongation factor, named Se1B. Its
amino-terminal three domains show homology to elongation factor EF-Tu
and accordingly bind GTP and selenocysteyl-tRNA(Sec), In addition, Se1B
exhibits a long carboxy-terminal extension that interacts with a
secondary structure of selenoprotein mRNAs (SECIS element) positioned
immediately downstream of the in-frame UGA codons specifying the sites
of selenocysteine insertion. In this report, a fast and efficient method
for the purification of large amounts of hexahistidine-tagged Se1B is
presented. After two chromatographic steps, 10 mg pure protein was
isolated from 12 g wet cell pellet. Biochemical analysis of the purified
protein showed that the tag does not influence the interaction of Se1B
with guanine nucleotides, SECIS elements, and selenocysteyl-tRNA(Sec).
In addition, the fusion protein is fully functional in mediating UGA
read-through in vivo. It therefore represents an excellent model for
studying the-function of Se1B and the mechanisms of selenocysteine
incorporation. (C) 2003 Elsevier Science (USA). All rights reserved.
