Novel Amino Acids Can Be Inserted at Certain Stop Codons

KEY CONCEPTS
- The insertion of selenocysteine at some UGA codons requires the action of an unusual tRNA in combination with several proteins.
- The unusual amino acid pyrrolysine can be inserted at certain UAG codons.
- The UGA codon specifies both selenocysteine and cysteine in the ciliate Euplotes crassus.
At least two known instances have been identified in which a stop codon is used to specify an unusual amino acid other than the Ile standard 20. Only particular stop codons are reinterpreted in this way by the translational apparatus. This demonstrates that the meaning of the codon triplet is influenced by the identity of other bases in the mRNA. Such a dual meaning for a particular codon in a genome should be distinguished from the context-independent complete reassignment of codons in some organisms or in mitochondria, as described in the previous section, The Universal Code Has Experienced Sporadic Alterations.
Selenocysteine, in which the sulfur of cysteine is replaced by selenium, is incorporated at certain UGA codons within genes coding for selenoproteins in all three domains of life. Usually, these proteins catalyze oxidation-reduction reactions. The selenocysteine residue is typically located in the active site, where it directly facilitates the reaction chemistry. For example, the UGA codon specifies selenocysteine in three E. coli genes encoding formate dehydrogenase isozymes; the incorporated selenium directly ligates a catalytic molybdenum ion in the active site.
Organisms capable of encoding selenocysteine possess an unusual tRNA, tRNA^Sec , which is more than 90 nucleotides long and contains acceptor and T stems of nonstandard length. Instead of seven base pairs in the acceptor stem and five in the T stem (a 7/5 structure), bacterial tRNA^Sec possesses an 8/5 structure, and archaeal and eukaryotic tRNA^Sec likely possess a 9/4 structure. These tRNAs also possess the 5′-UCA anticodon, allowing them to read UGA. In all organisms, tRNA^Sec is first aminoacylated with serine by seryl-tRNA^Sec synthetase (SerRS) to produce seryltRNA^Sec. In bacteria, the enzyme selenocysteine synthase next converts Ser-tRNA^Sec directly to selenocysteinyl (Sec)-tRNA^Sec using selenophosphate as the selenium donor. In archaea and eukaryotes, Ser-tRNA^Sec is first phosphorylated by the kinase PSTK to produce phosphoseryl (Sep)-tRNA^Sec . In a second step,
Sep-tRNA^Sec is converted to Sec-tRNA^Sec by the enzyme SepSecS. The exquisite specificity of PSTK is notable: It is capable of efficiently phosphorylating Ser-tRNA^Sec while excluding the standard Ser-tRNA^Ser . Improper phosphorylation of Ser-tRNA^Ser by PSTK could result in the incorporation of selenocysteine in response to serine codons.
The choice of which UGA codons are to be interpreted as selenocysteine is determined by the local secondary structure of the mRNA. A hairpin loop downstream of the UGA codon, termed the SECIS element, is required for incorporation of selenocysteine and exclusion of release-factor binding. The SECIS element is directly adjacent to the UGA codon in bacteria but is located in the 3′ untranslated region (UTR) of the mRNA in archaea and eukaryotes. In E. coli, a specialized translation elongation factor, SelB, interacts solely with Sec-tRNA^Sec and not with any other aminoacylated tRNA, including the precursor Ser-tRNA . SelB also binds directly to the SECIS element. The consequence of the action of SelB is that only those UGA codons that also possess a properly juxtaposed SECIS site will be able to productively bind Sec-tRNA^Sec in the ribosomal A site (FIGURE 1). Archaea and eukaryotes possess a homolog to SelB but also require the presence of an additional protein, SBP2, to permit the ribosome to insert selenocysteine.

FIGURE 1.SelB is an elongation factor that specifically binds tRNA^Sec to a UGA codon that is followed by a stem-loop structure in mRNA.
Another example of the insertion of a special amino acid is the placement of pyrrolysine at certain UAG codons in the archaeal genus Methanosarcina as well as in a few bacteria. In Methanosarcina, pyrrolysine is found in the active site of methylamine methyltransferases, where it plays an important role in the reaction chemistry. The incorporation of pyrrolysine requires a specialized aminoacyl-tRNA synthetase, pyrrolysyl-tRNA synthetase (PylRS), which aminoacylates a specialized tRNA^Pyl with pyrrolysine. tRNA^Pyl possesses the 5′-CUA anticodon, enabling it to read UAG. As with tRNA^Sec , tRNA^Pyl also possesses unusual structural features not found in other tRNAs; for example, it lacks the otherwise invariant U8 nucleotide and features atypically short D-loops and variable loops. The mechanism by which particular UAG codons are read as pyrrolysine has not yet been resolved, because it has not been possible to unambiguously identify a secondary structure element in all mRNAs that incorporate the amino acid. Further, no specific elongation factor targeting PyltRNA^Pyl to the ribosome has been identified.
Recently, it was found that the UGA codon specifies insertion of either cysteine or selenocysteine in the ciliate E. crassus. Dual use of UGA was found to occur even within the same gene, and the choice of which amino acid is inserted depends on the structure of the 3′ untranslated region of the mRNA. UGA specifies Cys generally in Euplotes and does not function as a stop codon. As a result, this work shows that position-specific dual use can occur within the context of a codon that is not otherwise used for termination in that organism.