The direct genetic encoding of pyrrolysine

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Pyrrolysine is an amino acid encoded by the amber codon in genes required for methylamine utilization by members of the Methanosarcinaceae. Pyrrolysine and selenocysteine share the distinction of being the only two non-canonical amino acids that have entered natural genetic codes. Recent experiments have shown that encoding of pyrrolysine, unlike that of selenocysteine, also shares an important trait of the original set of twenty amino acids. UAG is translated as pyrrolysine with the participation of a dedicated aminoacyl-tRNA synthetase. Expression of the genes encoding the pyrrolysyl-tRNA synthetase and its cognate tRNA is sufficient to add pyrrolysine to the genetic code of a recombinant organism. Thus, the recruitment of pyrrolysine into the genetic code involved evolution of the first non-canonical aminoacyl-tRNA synthetase and cognate tRNA to be described from nature.

Introduction

As the first group of organisms identified by Woese to be phylogenetically distinct from the Bacteria and the Eucarya, the methanogens became the charter members of the Archaea [1]. Anticipation thus ran high that the biochemistry of methanogenesis would yield new insights, and methanogens did not disappoint. A unique set of unusual cofactors is required for methanogenesis [2, 3, 4, 5]. Nonetheless, methanogens exceeded expectations when certain types of methanogenesis were found to further depend on pyrrolysine; the 22nd genetically encoded amino acid to be found in nature [6, 7].

Most methanogens can convert CO2 to methane. Members of the Methanosarcinacea, however, have also evolved methanogenic pathways for CO, acetate, methanol, methylthiols and methylamines [8, 9, 10••]. Pyrrolysine appears to have been retained in the genetic code of these methanogens for the synthesis of the enzymes that initiate methanogenesis from methylamines.

This brief review will outline the relationship between methylamine-dependent methanogenesis and amber codon translation as pyrrolysine; will describe how UAG translation appears to occur in the light of current data; and will discuss the implications that pyrrolysine holds for the detection of further additions to the genetic code in nature.

Section snippets

Methyltransferase genes with an amber sense codon

Methylation of coenzyme M (CoM) is the penultimate step in the formation of methane from methylamines [2, 3]. Methyl-CoM is disproportionated to methane and carbon dioxide, generating both energy and major anabolic precursors. The three methyltransferases MttB, MtbB and MtmB that initiate CoM methylation from trimethylamine, dimethylamine and monomethylamine, respectively, are highly abundant cellular proteins [11, 12, 13]. Each methyltransferase specifically methylates a cognate corrinoid

Structure and proposed function of pyrrolysine

Following their discovery, it was speculated that the amber codon of methylamine methyltransferase genes could encode a specialized amino acid important for methyltransferase function [16, 17]. Transcriptional alternatives, such as editing or intron processing, were eliminated by sequencing and S1 nuclease analysis of methylamine methyltransferase transcripts [16]. Translational alternatives, such as ribosome hopping, were eliminated by peptide sequencing of MtmB, which indicated the UAG codon

20, 21, 22… 21?

Pyrrolysine was designated the 22nd genetically encoded amino acid [25], following as it did after selenocysteine, the first genetically encoded amino acid described beyond the original twenty [26]. Selenocysteine had previously served as the only model for how a non-canonical amino acid could be incorporated into the genetic code. Pyrrolysine and selenocysteine do share certain traits: for example, both amino acids are encoded by a canonical stop codon (UGA for selenocysteine), and dedicated

tRNACUA aka tRNAPyl

The pyrrolysine tRNAPyl, also known as tRNACUA, is encoded by the pylT gene adjacent to the mtmB1 gene cluster in members of the Methanosarcinacea [7]. The D. hafniense genome has a pylT homolog, as well as an mttB homolog with an in-frame amber codon [7]. The tRNAPyl sequences from Methanosarcinaceae are highly conserved, but are much more diverged in D. hafniense, indicating potentially important bases and structural features that might be significant for charging with pyrrolysine (Figure 2).

The direct genetic encoding of pyrrolysine

UAG decoding as pyrrolysine is achieved by the charging of pyrrolysine onto tRNAPyl by pyrrolysyl-tRNA synthetase, PylS. PylS is the product of the pylS gene adjacent to pylT [7]. PylS was initially reported to be a lysyl-tRNA synthetase, but this activity might have been as a result of misfolding of an unstable form of recombinant PylS [7, 29••]. By contrast, PylS with a carboxy-terminal His tag is stable and does not recognize any standard amino acid. Instead, it can activate synthetic

Adding pyrrolysine to the genetic code of E. coli

Amber suppression can be used to introduce novel amino acids into the genetic code of Escherichia coli that carry an aminoacyl-tRNA synthetase mutated to charge an amber suppressor tRNA with an unnatural amino acid [35]. Similarly, E. coli bearing both pylT and pylS translates the amber codon within recombinant mtmB1 transcripts [29••] using exogenously supplied synthetic pyrrolysine [23••].

These experiments were a robust confirmation of the in vitro charging experiments and further illustrate

A potential lysylation pathway

Prior to the discovery of the pyrrolysyl-tRNA synthetase, class 1 and class 2 lysyl-tRNA synthetases of M. barkeri were observed to jointly carry out the charging of tRNAPyl with lysine in vitro [32]. The physiological role of this activity is yet unknown. It seems unlikely to be the primary pathway of pyrrolysyl-tRNAPyl formation in the organism, given its slow rate (approximately 1 turnover/hour) and the ability of PylS to charge tRNAPyl with pyrrolysine in vivo and in vitro. Nonetheless, this

Trans- and cis-acting factors in UAG translation

Previously, in vitro experiments had shown that elongation factor EF-Tu binds lysyl-tRNAPyl [33••]. The translation of UAG as pyrrolysine within E. coli indicates that pyrrolysyl-tRNAPyl is also recognized by EF-Tu [29••]. By contrast, selenocysteinyl-tRNASec is not recognized by EF-Tu, but by specialized elongation factors (SelB or EFSec). In Bacteria, SelB binds to a transcript structural element (SECIS) immediately 3′ of the UGA codon promoting UGA translation, rather than termination [26].

Conclusions — 20, 21, 22…23?

Shortly before the discovery of pyrrolysine was reported, Balakrishnan published a group theoretical analysis of the existent genetic code, and predicted that UAG would be found to encode a novel amino acid similar to histidine [43]. Although one can debate the extent to which histidine and pyrrolysine are similar, it is certainly true that their 5-member rings both possess nitrogens that are ionizable in the physiological range. The analysis went on to predict that UAA would be found to encode

Update

We now have an article in press [46••] that describes a mutant that is unable to translate UAG as pyrrolysine because of a deletion of pylT and of its promoter. This mutant grows well on all substrates except on methylamines, which indicates that the major role of pyrrolysine in Methanosarcina spp. is methylamine metabolism. In addition, a bioinformatics study has been released [47••] that provides a new algorithm for detection of pyrrolysyl-proteins. A very small number of proteins could be

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Work in the author's laboratory was supported from funds from NSF (MCB-9808914), DOE (DEFG02-92ER20042) and NIH (GM061796 and GM070663).

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