Transfer RNAs with novel cloverleaf structures

Abstract

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

A Facile Method for Producing Selenocysteine‐Containing Proteins

“…We designed allo-tRNA UTu2D (Figure 3A and Figure S6) from an allo-tRNA Ala species that carries a different cloverleaf structure to allo-tRNA UTu1. [15] Surprisingly, GPx1 proteins produced with allo-tRNA UTu1D and allo-tRNA UTu2D exhibited similar glutathione peroxidase (GPx) activities (Figure 3B), which is also supported by mass spectrometry data (Figure S7). Because allo-tRNA UTu2D expressed from a weak tRNA promoter was more easily sequestered by excess SelA than allo-tRNA UTu1D (Figure 3C), the former tRNA required a smaller amount of As SelA for Sec-tRNA formation.…”

Rewriting the Genetic Code

“…Given the vast amount of new genomic and metagenomic DNA sequence information generated in the past few years, we document here the current knowledge of natural deviations from the standard genetic code (45, 61, 79, 92, 94, 98, 115, 125, 134, 157) (Figure 2) (Supplemental Figure 1). Previously, it was well known that Candida yeast reassigns the CUG codon from leucine (Leu) to serine (Ser) (60) and that many ciliates reassign either the UGA stop codon or both the UAA and the UAG stop codons to a canonical amino acid (12, 61, 88, 119) (Figure 2).…”

“…In particular, all Geodermatophilaceae (actinobacteria) species sequenced so far use UAG for Sec (Figure 2). In contrast, it was predicted that the UGA stop codon would be recoded as both Sec and Cys in a few Deltaproteobacteria species such as Desulfococcus biacutus (98), although experimental validation is required (Figure 2). Furthermore, a group of potential missense and nonsense suppressor tRNA genes was identified in genome/metagenome/metatranscriptome sequences, derived mostly from Acidobacteria species (98), indicating that ambiguous decoding of a particular codon might be a popular mechanism in bacteria.…”

Eine einfache Methode zur Produktion von Selenoproteinen

“…[15] Einige dieser allo-tRNAs fungieren in E. coli als tRNA Ser,[15] und wir vermuteten, dass sie möglicherweise auch Erkennungssequenzen für SelA enthalten (Abbildung 1 A). Dabei zeigten einige der per Metagenomanalyse identifizierten allo-tRNA-Spezies (allo-tRNA UTu1 ) eine ähnliche Ambersuppressionseffizienz wie E.-coli-supD -tRNA Ser (Abbildung 1 A und Hintergrundinformationen, Abbildung S1), die eine der aktivsten Suppressor-tRNAs in E. coli ist.…”

“…Diese beruhte auf allo-tRNA Ala und weist eine von allo-tRNA UTu1 abweichende Kleeblattstruktur auf. [15] Überraschenderweise zeigten GPx1-Proteine, die mit allot-RNA UTu1D oder allo-tRNA UTu2D produziert wurden, ähnliche Glutathionperoxidase-Aktivität (Abbildung 3 B); ähnliche Raten für den Sec-Einbau in die GPx1-Proteine wurden außerdem per Massenspektrometrie bestätigt (Abbildung S7). Weil allo-tRNA UTu2D mithilfe eines schwachen tRNA-Promotors exprimiert wurde, konnte sie höchstwahrscheinlich einfacher von überschüssigem SelA gebunden werden als allo-tRNA UTu1D (Abbildung 3 C).…”