Partitioning between recoding and termination at a stop codon-selenocysteine insertion sequence.

Suresh Babu Kotini,Marina V Rodnina,Frank Peske

doi:10.1093/nar/gkv558

Suresh Babu Kotini, Marina V Rodnina + Show 1 more

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https://doi.org/10.1093/nar/gkv558

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Abstract

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNASec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.

Highlights

Selenocysteine (Sec) is the 21st genetically encoded amino acid that is incorporated into proteins during protein synthesis on the ribosome [1,2]
The most plausible reason for the difference in the reported values is the low concentration of selenium (1 ␮M) used in the earlier work, which appears too low compared to the concentration required to reach the maximum of Sec incorporation in our experiments (>10 ␮M Na2SeO3)
We note that such high selenium concentrations are probably not needed for the synthesis of the endogenous bacterial selenoproteins; the optimal concentration may depend on the reporter construct used

Summary

Introduction

Selenocysteine (Sec) is the 21st genetically encoded amino acid that is incorporated into proteins during protein synthesis on the ribosome [1,2]. Selenoproteins are found in bacteria, archaea and many eukaryotes [3,4,5,6,7]. Sec is structurally similar to cysteine (Cys), with a selenium atom at the place of the sulfur, forming a selenol group. Unlike the 20 standard amino acids which are encoded by their specific sense codons, Sec is encoded by a stop codon, UGA, which normally serves as a signal for the termination of protein synthesis. The specific incorporation of Sec into proteins requires a selenocysteine insertion sequence

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