Molecular mechanism of selenocysteine incorporation in bacterial translation

Molecular mechanism of selenocysteine incorporation in bacterial translation

English

Suresh Babu Kotini

Doctoral thesis

Date of Examination: 2011-06-27

Date of issue: 2012-06-27

Advisor: Rodnina, Marina Prof. Dr.

Referee: Rodnina, Marina Prof. Dr.

Referee: Stark, Holger Prof. Dr.

Referee: Ficner, Ralf Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-000D-F0BB-9

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Abstract

English

Selenocysteine is the 21st amino acid which is incorporated into proteins by recoding a stop codon UGA followed by a selenocysteine insertion sequence (SECIS) of the mRNA. In bacteria, selenocysteine insertion requires specialized machinery which includes selenocysteine-specific tRNASec, translation factor SelB which delivers Sec-tRNASec to the ribosome, as well as proteins SelA, SelD, and seryl-tRNA synthetase which are required to produce Sec-tRNASec. The aim of this work is to develop experimental assays to study Sec incorporation into proteins in vivo and in vitro. As an in vivo assay, we designed the dual luciferase reporter assay and validated its performance using Western blots and luciferase reactions. The efficiency of Sec incorporation was 35% independent of growth conditions. Rapidly growing cells required additional selenium source for efficient Sec insertion. This level of UGA recoding could be reproduced in the fully reconstituted in vitro translation system upon synthesis of a fragment of a natural selenoprotein FdfH. The recruitment of SelB to the SECIS-element prior to translation prevented inhibition of Sec insertion by RF2, a termination factor which usually recognizes the UGA codon and competes with Sec-tRNASec for binding to the ribosome. These results shed light on the importance of the SECIS and on the mechanism by which a stop codon is redirected for efficient readthrough by a specific tRNA.

Keywords: selenocysteine; SECIS-element; in vivo; in vitro; translation; ribosome; selB