Formation of a Ternary Complex for Selenocysteine Biosynthesis in Bacteria

Ivan R Silva,Vitor H.B Serrão,Livia R Manzine,Lívia M Faim,Marco T.A Da Silva,Raphaela Makki,Daniel M Saidemberg,Marinônio L Cornélio,Mário S Palma,Otavio H Thiemann

doi:10.1074/jbc.m114.613406

Abstract

The synthesis of selenocysteine-containing proteins (selenoproteins) involves the interaction of selenocysteine synthase (SelA), tRNA (tRNA(Sec)), selenophosphate synthetase (SelD, SPS), a specific elongation factor (SelB), and a specific mRNA sequence known as selenocysteine insertion sequence (SECIS). Because selenium compounds are highly toxic in the cellular environment, the association of selenium with proteins throughout its metabolism is essential for cell survival. In this study, we demonstrate the interaction of SPS with the SelA-tRNA(Sec) complex, resulting in a 1.3-MDa ternary complex of 27.0 ± 0.5 nm in diameter and 4.02 ± 0.05 nm in height. To assemble the ternary complex, SPS undergoes a conformational change. We demonstrated that the glycine-rich N-terminal region of SPS is crucial for the SelA-tRNA(Sec)-SPS interaction and selenoprotein biosynthesis, as revealed by functional complementation experiments. Taken together, our results provide new insights into selenoprotein biosynthesis, demonstrating for the first time the formation of the functional ternary SelA-tRNA(Sec)-SPS complex. We propose that this complex is necessary for proper selenocysteine synthesis and may be involved in avoiding the cellular toxicity of selenium compounds.

Highlights

Selenoprotein biosynthesis requires the interaction of tRNASec and specific enzymes that drive the synthesis of selenocysteine
Sec biosynthesis in E. coli requires 10 molecules of SertRNASec covalently bound to homodecameric SelA to catalyze the conversion of Ser to Sec (5)
Homodecamerization, and the Ser-Sec conversion and selenoprotein biosynthesis, is dependent on the N-terminal region, as we observed by functional complementation with the N-terminally truncated E. coli SelA

Summary

Background

Selenoprotein biosynthesis requires the interaction of tRNASec and specific enzymes that drive the synthesis of selenocysteine. Our results provide new insights into selenoprotein biosynthesis, demonstrating for the first time the formation of the functional ternary SelAtRNASec-SPS complex. We propose that this complex is necessary for proper selenocysteine synthesis and may be involved in avoiding the cellular toxicity of selenium compounds. Sec is synthesized on the specific L-serine-aminoacylated tRNA (Ser-tRNASec) and incorporated into selenoproteins at UGA codons via a complex pathway that works through transient protein-RNA and protein-protein interactions. In bacteria, this pathway requires the specific tRNASec (SelC) and an mRNA-specific structure called selenocysteine insertion sequence (SECIS) (1, 3). The structures of Aquifex aeolicus SelA and its binary complex SelA-tRNASec

The abbreviations used are

Experimental Procedures

Results

Discussion