Mutagenesis of selC , the gene for the selenocysteine-insertlng tRNA-species in E.coli : effects on in vivo function (original) (raw)

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Lehrstuhl für Mikrobiologie der Universität München

Maria-Ward-StraBe 1a, D-8000 Munich 19, FRG

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,

Lehrstuhl für Mikrobiologie der Universität München

Maria-Ward-StraBe 1a, D-8000 Munich 19, FRG

Search for other works by this author on:

Lehrstuhl für Mikrobiologie der Universität München

Maria-Ward-StraBe 1a, D-8000 Munich 19, FRG

* To whom correspondence should be addressed

Search for other works by this author on:

Published:

01 January 1990

Received:

08 October 1990

Accepted:

31 October 1990

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Christian Baron, Johann Heider, August Böck, Mutagenesis of selC , the gene for the selenocysteine-insertlng tRNA-species in E.coli : effects on in vivo function , Nucleic Acids Research, Volume 18, Issue 23, 1 December 1990, Pages 6761–6766, https://doi.org/10.1093/nar/18.23.6761
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Abstract

The selenocysteine-inserting tRNA (tRNA Sec ) of E. coli differs In a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA Sec ; (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA 140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA 140 of the fdhF mRNA was replaced by a serine codon (UC-A). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA Sec which enable decoding of UGA 140 in the fdhF message.

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© 1990 Oxford University Press

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