Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA

Abstract

Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNA(CUA) with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.

Effects of Nitrogen and Carbon Sources on Transcription of Soluble Methyltransferases in Methanosarcina mazei Strain Gö1

“…This analysis showed that the observed changes in expression of the mtmBC operons during lag-or stationary-phase growth compared to exponential- Transcription of pyl genes does not appear to be affected by nitrogen limitation and the presence of TMA. All methylamine methyltransferase transcripts possess an internal amber codon (UAG) (15), which encodes the recently discovered amino acid pyrrolysine (12,19). Thus, synthesis of functional methylamine methyltransferases is dependent on the expression of the pyl genes, whose gene products are responsible for the synthesis of pyrrolysyl-tRNA Pyl .…”

“…Within this nomenclature, B describes the substrate specific methyltransferase, C the corrinoid binding polypeptide, and A the CoM-methylating protein. For M. barkeri multiple and nearly identical copies of the operons encoding DMA and MMA methyltransferases and their respective corrinoid proteins have been identified (15,19). Analyzing the genome sequence of M. mazei, seven operons coding for methylamine methyltransferases and their corresponding corrinoid proteins and three genes coding for methylcobalamine:CoM methyltransferases have been identified (4), an overview of which is depicted in Fig.…”

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

“…This new amino acid is co-translationally inserted in response to an in-frame UAG codon located in the corresponding mRNAs [3]. The insertion of this amino acid relies on the presence of a specific suppressor tRNA (tRNA Pyl ) [4] and the new class II aminoacyl-tRNA synthetase, pyrrolysyl-tRNA synthetase (PylRS) specific only for its substrates tRNA Pyl and Pyl [5,6]. The mechanism of Pyl-tRNA Pyl insertion at UAG is unknown, however, it has been proposed that a specialized pyrrolysine insertion element (PYLIS) is present immediately downstream of the UAG which assists in the recoding event.…”

Aminoacyl-tRNA synthetases: Versatile players in the changing theater of translation

“…In contrast to the widespread occurrence of selenocysteine, the distribution of pyrrolysine is so far known only in a handful of methanogenic archaea and bacteria (Galagan et al+, 2002;Srinivasan et al+, 2002)+ The central metabolism of these organisms is the catabolic conversion of methylamines, which requires a family of mono-, di-, and trimethylamine methyltransferases+ Many of the methyltransferase genes of the methanogen Methanosarcina barkeri are interrupted by in-frame amber codons; tryptic peptide sequencing of one such protein (MtmB) showed that the amber codon is decoded as lysine (James et al+, 2001)+ The determination of the X-ray structure of MtmB to a resolution of 1+55 Å showed additional electron density best described as a lysine in an amide linkage to a 4-substitutedpyrroline-5-carboxylate (Hao et al+, 2002)+ This unusual amino acid suggests a plausible (though as yet unproven) mechanism for the methyltransferase reaction (Hao et al+, 2002)+ The machinery necessary for pyrrolysine insertion in M. barkeri appears to reside in a unique gene cluster that includes an amber suppressing tRNA (pylT ), an unusual lysyl-tRNA synthetase (pylS ), and additional proteins provisionally responsible for converting lysine to pyrrolysine (Srinivasan et al+, 2002)+ The tRNA encoded by pylT differs significantly from canonical tRNAs+ The anticodon arm has 6 rather than 5 bp, the variable loop has 3 rather than 4 nt, and the nearly universally conserved D-loop GG and T-loop TcC sequences are absent+ These features are likely to be important for recognition by the dedicated pylS gene product, which resembles the class II tRNA synthetases in its C-terminal catalytic domain+ However, the N-terminal domain of the pylS lysyl-tRNA synthetase possesses negligible sequence identity with the anticodon binding domains of other class IIb synthetases, which are based on the OB fold+ An in-frame amber codon is also found in the mttB gene of the gram-positive bacterium Desulfitobacterium hafniense, and it is likely decoded as pyrrolysine by a cotranslational mechanism similar to that of selenocysteine+ In D. hafniense, however, pylS is split into two ORFs that separately encode the N-and C-terminal domains+ The presence of an unusual LysRS for pyrrolysine suggests that tRNA Lys(Pyl) has diverged beyond the point of recognition by LysRS-I or LysRS-II+ Features of the translation machinery that allow context dependent insertion of pyrrolysine remain to be identified, and constitute an interesting follow-up question+…”

“…Accurate translation of the genetic information into proteins is a complex ensemble performance by essential cellular players: the ribosome, messenger RNAs, aminoacylated tRNAs, and a host of additional protein and RNA factors+ Among the latter are the aminoacyl-tRNA synthetases (aaRS 1 ), which join amino acids with their cognate transfer RNAs in a high-fidelity reaction+ Although the principal functions of the aaRS in translation were established decades ago, these enzymes have continued to surprise us with their idiosyncratic origins, mechanistic complexities, and unexpected connections to other critical aspects of cellular function+ Like a venerable character actor playing against type in a new production, the aaRS and their close relatives are emerging with new functions in biology+ These include direct participation in amino acid biosynthesis, DNA replication, RNA splicing, and aspects of eukaryotic cell biology related to cytokine function and cell cycle control+ Many of these roles were discussed at the Fourth International Conference on Aminoacyl-tRNA Synthetases in Biology, Medicine, and Evolution, which was organized by the authors and held earlier this year at Asilomar+ The remarkable functional diversity of tRNA synthetases hints at the underlying flexibility and adaptability of the translation apparatus, a feature also highlighted by the recent report of a new amino acid, pyrrolysine (Hao et al+, 2002;Srinivasan et al+, 2002)+ This "22nd amino acid" is likely incorporated into proteins by use of the same strategy employed for the "21st amino acid," selenocysteine+ Here we summarize recent findings that strengthen our understanding of the catalytic mechanisms and substrate recognition properties of tRNA synthetases, particularly with regard to induced-fit conformational changes and amino acid editing+ We also describe new and highly significant developments in the field+ The emerging picture is of a family of enzymes distinguished by a multiplicity of biological roles, potential for impact in the evolution of biotechnology, and the ancient function in translation that sheds light on the molecular evolution of life (Fig+ 1)+ Readers should be advised that space limitations pre-clude us from discussing here many of the interesting program areas covered at the meeting, including aaRS-tRNA interactions, aaRS as therapeutic targets, aaRS evolution and phylogenetics, and other aspects of tRNA synthetase structure and function+ All aaRS catalyze a two-step aminoacylation reaction+ This entails condensation of the amino acid with ATP to form an activated aminoacyl adenylate intermediate, followed by transfer of the amino acid to the 39-terminal ribose of tRNA to generate the aminoacylated product (Ibba & Soll, 2000)+ In this way, each amino acid becomes associated with one or more anticodon sequences in the cognate tRNA isoacceptor set, and thus a corresponding set of codons+ In the original adaptor hypothesis proposed by Francis Crick, each amino acid is associated with its unique cognate aaRS, such that a typical cell would possess a full complement of 20 different aaRS to accommodate all of the standard amino acids used in translation (Crick, 1958)+ A major insight gained in the last decade has been that these 20 canonical enzymes are divided evenly into two classes, each of which represents a distinct evolutionary solution to the requirement for the aminoacylation reaction+ Enzymes of the same family share a characteristic catalytic fold, identifiable peptide sequence motifs, and distinctive mechanistic features (Ibba & Soll, 2000)+ Thus, the catalytic domains of class I enzymes are based on a Rossmann dinucleotide binding fold, whereas those of class II enzymes are organized around a si...…”