Rewiring Translation for Elongation Factor Tu‐Dependent Selenocysteine Incorporation

Abstract

Selenium is an essential micronutrient for animals. [1] Humans contain 25 presumably essential selenoproteins [2] in which selenium is found in the form of Sec. [3] In this 21 st genetically encoded amino acid [4] the thiol moiety of Cys is replaced by a selenol group. In all Sec-decoding organisms, Sec biosynthesis (Scheme 1B) starts with the acylation of tRNA Sec by seryl-tRNA synthetase (SerRS) to form Ser-tRNA Sec (reviewed in [5]). In bacteria, conversion of Ser-tRNA Sec to Sec-tRNA Sec is achieved by SelA (reviewed in [4]). In contrast, archaea and eukaryotes employ an additional phosphorylation step. Ophosphoseryl-tRNA Sec kinase (PSTK) phosphorylates the tRNA-bound Ser moiety of Ser-tRNA Sec to form O-phosphoseryl-tRNA Sec (Sep-tRNA Sec), [6] the substrate for SepSecS that catalyzes the tRNA-dependent Sep to Sec conversion. [7] The selenium donor for both SelA and SepSecS is selenophosphate (reviewed in [4, 7b]). During selenoprotein synthesis Sec is co-translationally incorporated by a reprogrammed UGA stop codon. A specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element) located within the bacterial ORF sequence are required for unambiguous Stop to Sec recoding. [4] EF-Tu does not recognize Sec-tRNA Sec and also discriminates against Ser-tRNA Sec.[4] Selenium and sulfur are in the same group of elements in the periodic table and share certain properties (e.g., size, electronegativity, major oxidation states); yet, Cys and Sec are distinguished by different electrode potentials, [8] nucleophilicity (Cys < Sec), [9] and sidechain pK a (8.3 for Cys vs 5.2 for Sec). [10] Thus, selenoproteins have unique properties [11]. Sec is frequently found as an enzyme active site residue endowing these proteins (e.g., redox enzymes) with superior catalytic activities. Sec to Cys replacements in selenoenzymes may lead to 10 to 1,000-fold activity loss (reviewed in [11b]). While disulfides occur frequently in proteins to increase stability or provide redox functions, diselenides are much less frequent. [12] The occurrence of diselenides in proteins has exciting biological and biomedical significance, as they are more stable than disulfides [13] and sometimes even resistant to reduction by DTT. [12]

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

“…Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3,4), aminoacyl-tRNA synthetases (AARSs) (5,6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).…”

A synthetic tRNA for EF‐Tu mediated selenocysteine incorporation in vivo and in vitro

“…To characterize in vitro formation of Sec‐tRNA, tRNA species were radiolabeled using [α‐ 32 P]ATP and the E. coli CCA editing enzyme [11]. Ser‐tRNA formation by SerRS, selenophosphate production by SelD, and Ser to Sec conversion by SelA was carried out under anoxic conditions as previously described [8]. Conversion rates were determined by autoradiography and by quantitation of aminoacyl‐AMP after thin layer chromatography of nuclease P1 digests of aminoacyl‐tRNA UTu [12].…”

“…Therefore, if the SelA‐dependent conversion of Ser‐tRNA UTu to Sec‐tRNA UTu is not complete, Ser will be incorporated instead of the desired Sec residue. This was an impediment in the earlier work in which ∼30% misincorporation of Ser was observed [8]. We reasoned that by designing an improved tRNA UTu with better substrate properties for SelA, misincorporation could be prevented.…”

“…In bacteria, the SECIS sequence is located directly after the suppressed UGA and is thus part of the coding sequence of bacterial genes, making engineering of newly designed selenoproteins very difficult [5–7]. Recently, we reported construction of a synthetic tRNA (tRNA UTu ) that enabled SECIS‐independent and EF‐Tu‐dependent insertion of Sec in Escherichia coli [8]. This tRNA UTu combines the aminoacyl acceptor helix of tRNA Sec with the backbone of tRNA Ser , and serves as a substrate for the essential proteins SerRS, SelA, and EF‐Tu.…”

Umkodierung des genetischen Codes mit Selenocystein

“…Jede FDH H -Genvariante (fdhF 140 NNN) wurde zusammen mit selA, selB und den selC-Mutanten (tRNA Sec NNN ), welche das entsprechende Anticodon trugen, in einem E.coli-DselADselBDfdhF-Deletionsstamm (MH5) [12] Während sich mithilfe des FDH H -in-vivo-Aktivitätstests qualitativ zeigen ließ, dass der Sec-Apparat in der Lage ist, nahezu alle Codons zu Sec zu rekodieren (Abbildung 1), wurden In-vitro-BV-Reduktionstests mit allen 64 unter anaeroben Bedingungen gereinigten, rekombinanten FDH H -Varianten durchgeführt, um den Grad an Sec-Einbau zu quantifizieren (Abbildungen S1, S2 Vierzig weitere Codons zeigten eine weniger eindeutige Umkodierung an Position 140 von FDH H . Dreißig hiervon (Gruppe III) ermçglichten die Umwandlung der ursprünglichen Bedeutung zu Sec mit 12-72 % Effektivität, wie anhand der spezifischen BV-Reduktion ermittelt wurde (Abbildung 2, Tabelle S1).…”