Molecular and General Genetics MGG

, Volume 262, Issue 4–5, pp 800–806

Genetic probing of the interaction between the translation factor SelB and its mRNA binding element in Escherichia coli

  • M. Kromayer
  • B. Neuhierl
  • A. Friebel
  • A. Böck
ORIGINAL PAPER

DOI: 10.1007/s004380051143

Cite this article as:
Kromayer, M., Neuhierl, B., Friebel, A. et al. Mol Gen Genet (1999) 262: 800. doi:10.1007/s004380051143

Abstract

Decoding of the UGA codon in mRNAs for selenoproteins as selenocysteine requires interaction of the translation factor SelB with an mRNA structure, the SECIS element. A genetic analysis of this interaction was performed by selecting for intergenic suppressor mutations in selB which counteracted the detrimental effect of defined mutations in the SECIS element. Both allele-nonspecific and allele-specific mutations, as judged by readthrough of the UGA into the LacZ-encoding segment of fdhF ′-′lacZ fusions and by incorporation of selenium, were isolated. selB genes from ten suppressor mutants were sequenced and the corresponding mutations were localized to five positions within the protein. Four of the suppressors had amino acid exchanges within a 23-amino acid stretch in domain 4b of SelB, which probably represent sites of contact between the protein and the mRNA. A fifth mutation was localized in domain 4a of SelB; it promoted allele-nonspecific readthrough. Since a truncated SelB species lacking domain 4b did not show complex formation with the SECIS element, we speculate that the latter mutation affects the interaction between the tRNA-binding and the mRNA-binding domains. None of the SelB variants was able to promote UGA readthrough when major structural changes that altered the length of the helical part or enlarged the apical loop were introduced into the SECIS element. The results obtained also show that novel pairs of SelB/SECIS derivatives can be generated which may be useful for the targeted insertion of selenocysteine into proteins.

Key words Selenocysteine SelB SECIS element Intergenic suppressors 

Copyright information

© Springer-Verlag Berlin Heidelberg 1999

Authors and Affiliations

  • M. Kromayer
    • 1
  • B. Neuhierl
    • 1
  • A. Friebel
    • 1
  • A. Böck
    • 1
  1. 1.Institut für Genetik und Mikrobiologie Lehrstuhl für Mikrobiologie der Universität München Maria-Ward-Strasse 1a, D-80638 München, Germany e-mail: august.boeck@lrz.uni-muenchen.de Fax: +49-89-21806122DE

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