In vivo probing of SECIS-dependent selenocysteine translation in Archaea.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure-function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial ß-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Sulphate assimilation under Cd2+ stress in Physcomitrella patens--combined transcript, enzyme and metabolite profiling.

Cd(2+) causes disturbance of metabolic pathways through severe damage on several levels. Here we present a comprehensive study of Cd(2+)-mediated effects on transcript, enzyme and metabolite levels in a plant without phytochelatin (PC). The moss Physcomitrella patens (Hedw.) B.S.G. was stressed with up to 10 microm Cd(2+) to investigate the regulation of gene transcription and activities of enzymes involved in the assimilatory sulphate reduction pathway and in glutathione biosynthesis. Real-time PCR, specific enzyme assays as well as thiol peptide profiling techniques were applied. Upon supplementation of 10 microm Cd(2+), the moss showed a more than fourfold increase in expression of genes encoding ATP sulphurylase (ATPS), adenosylphosphosulphate reductase, phosphoradenosylphosphorsulphate reductase, sulphite reductase (SiR) and gamma-glutamyl cysteine synthetase (gamma-ECS). Likewise, elevated enzyme activities of gamma-ECS and glutathione synthetase were observed. Contrarily, activity of O-acetylserine (thiol) lyase (OAS-TL), responsible for biosynthesis of cysteine, was diminished. At the metabolite level, nearly doubling of intracellular cysteine and glutathione content was noted, while the moss did not produce any detectable amounts of PCs. These results suggest a Cd(2+)-induced activation of the assimilatory sulphate reduction pathway as well as of glutathione biosynthesis on different levels of regulation.

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs.

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.