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Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNAPyl). The secondary structure of tRNAPyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNAPyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNAPyl, its interaction with PylRS, and survey recent progress on the use of tRNAPyl as a tool for genetic code expansion.

The tRNA for the 21st proteinogenic amino acid, selenocysteine, exists in mammalian cells as 2 isoforms differing by a single 2'-O-methylribosyl moiety at position 34 (Um34). These isoforms contain either 5-methoxycarbonylmethyluridine (mcm5U) or 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) at position 34. The accumulation of the mcm5Um isoform is tightly correlated with the expression of nonessential "stress response" selenoproteins such as glutathione peroxidase 1 (GPX1). The expression of essential selenoproteins, such as thioredoxin reductase 1 (TXNRD1), is not affected by changes in Sec-tRNA[Ser]Sec isoform accumulation. In this work we used purified mcm5U and mcm5Um Sec-tRNA[Ser]Sec isoforms to analyze possible differences in binding to the selenocysteine-specific elongation factor, EEFSEC, and the translation of GPX1 and TXNRD1in vitro. Our results indicate that no major distinction between mcm5U and mcm5Um isoforms is made by the translation machinery, but a small consistent increase in GPX1 translation is associated with the mcm5Um isoform. These results implicate fundamental differences in translation efficiency in playing a role in regulating selenoprotein expression as a function of isoform accumulation.

The ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. Both ordered and intrinsically disordered proteins (IDPs) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. The exceptional structural plasticity of IDPs, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and IDPs. In fact, some residues (so-called disorder-promoting residues) are noticeably more common in IDPs than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. Furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue.