Key nucleotide sites of Escherichia coli tRNASec

Abstract:

In prokaryotes, selenoprotein synthesis requires the interaction between selenocysteine (Sec)-specific tRNA^Sec (SelC) and selenocysteinyl tRNA synthase (SelA) or Sec-specific elongation factor (SelB).[Objective] Based on the Sec insertion machinery in Escherichia coli, we aimed at finding key sites of tRNA^Sec scaffold and providing a new way to solve the problems of low Sec insertion efficiency and low yield of selenoprotein.[Methods] We used rat cytoplasmic thioredoxin reductase (thioredoxin reductase 1, TrxR1) as a model selenoprotein. First, we constructed tRNA^Sec by site-directed mutagenesis, and transformed them to BL21 (DE3) gor-to obtain a positive recombinant strain (carrying pET-TRSter'/pSUABC), which was used to express rat cytoplasmic thioredoxin reductase 1 (TrxR1). Then TrxR1 was purified using 2',5'-ADP Sepharose affinity chromatography and gel filtration. The enzyme activities of TrxR1 were determined by the classical Se-dependent DTNB reduction assay to analyze the key nucleotide sites and evaluate the Sec incorporation efficiency.[Results] When tRNA^Sec co-expressed with SelA and SelB in the presence of the bacterial SECIS element, the activities of TrxR1 decreased at varying degrees compared with wild type. Among them, the enzyme activities of all G18 and G19 mutants were much lower than that of wild type (<10%). However, the enzymatic activities of a26 and b7 were relatively high.[Conclusion] G18 and G19 nucleotide sites of E. coli tRNA^Sec may play a vital role in maintaining the stability and flexibility of tRNA^Sec. Site-directed mutations of tRNA^Sec causing the conformational change may affect the interaction between tRNA^Sec and Sec elements. Thus, it is possible to improve the Sec insertion efficiency via proper modification on tRNA nucleotide sites.

Key words:Escherichia coli;selenocysteine (Sec);selenoprotein;Sec incorporation tRNA^Sec;cytoplasmic thioredoxin reductase (TrxR1)