Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

Abstract

The genetic code, initially thought to be universal and immutable, is now known to contain many variations, including biased codon usage, codon reassignment, ambiguous decoding and recoding. As a result of recent advances in the areas of genome sequencing, biochemistry, bioinformatics and structural biology, our understanding of genetic code flexibility has advanced substantially in the past decade. In this Review, we highlight the prevalence, evolution and mechanistic basis of genetic code variations in microorganisms, and we discuss how this flexibility of the genetic code affects microbial physiology.

Figure 1. Mechanisms of genetic code flexibility

a ∣ Each amino acid is attached to the corresponding tRNA by a specialized aminoacyl-tRNA synthetase (aaRS), in a reaction called aminoacylation. For example, threonyl-tRNA synthetase (ThrRS) selects Thr out of the amino acid pool and ligates it on to the 3′ end of tRNA^Thr. The resulting aminoacyl-tRNAs are then delivered to the ribosome by initiation or elongation factors to decode the matching codon. b ∣ There are multiple mechanisms of genetic code flexibility. Codon bias refers to selective usage of synonymous codons to encode the same amino acid. The frequency of codons in a given organism typically matches the cellular abundance of the corresponding tRNA. Codon reassignment requires evolution of a new tRNA to decode sense codons with a new amino acid, or a new tRNA that can decode stop codons with an amino acid. Ambiguous decoding refers to simultaneous decoding of the same codon by two or more amino acids in one cellular compartment; this could be caused by recognition of the same tRNA by more than one aaRS, by misacylation of a tRNA or by ribosomal decoding errors. Recoding traditionally refers to partial codon reassignment that is context dependent. For instance, in certain bacteria and eukaryotes, a subset of UGA stop codons with a nearby selenocysteine (Sec) insertion sequence (SECIS) element, in the presence of SelB, are recoded to Sec, whereas other UGA stop codons retain their ability to signal translational termination.

Figure 2. Biased codon usage

a ∣ Biased codon usage occurs when synonymous codons are decoded by different tRNA isoacceptors. As some synonymous codons are used at higher frequencies than others, this leads to biased codon usage. b ∣ Although the optimized usage of synonymous codons increases the protein synthesis rate under most conditions, non-optimal codon usage can improve bacterial fitness under certain conditions. For example, codon optimization of the frq gene in Neurospora crassa results in a loss of fitness. frq controls the circadian clock function, and optimizing the codons of frq increases the expression of Frq but leads to defects in folding of this protein. These folding defects impair Frq function in the regulation of the circadian feedback loop (middle panel). Similarly, the kaiB and kaiC genes in the cold-adapted cyanobacterium Synechococcus elongatus are also enriched for non-optimal codons. The kaiB and kaiC genes are critical for regulation of circadian rhythms, and codon optimization increases the protein levels of KaiB and KaiC and prevents the circadian rhythm from switching off, resulting in loss of fitness by the cyanobacteria (bottom panel). Part b of the image adapted from REF. 144, Nature Publishing Group.

Figure 3. Codon reassignment

a ∣ CUN codons are read as Leu in the standard genetic code through the use of tRNA^Leu_UAG. However, in some microorganisms, CUN codons have been reassigned to Thr. b ∣ CUN reassignment in mitochondria involved several steps. In the mitochondrial genome of some Saccharomycetaceae species (for example, Candida albicans), the tRNA^His_GUG gene was duplicated. The CUN codons and a tRNA^Leu_UAG that decodes CUN then disappeared, leading to a reduced genetic code, as in Kluyveromyces lactis. In Saccharomyces cerevisiae, one copy of tRNA^His_GUG evolved to carry an anticodon UAG that reads CUN codons (tRNA^His_UAG). The threonyl-tRNA synthetase (ThrRS) co-evolved with the tRNA^His_UAG to recognize it as a tRNA^Thr_UAG. CUN codons then reappeared to complete the codon reassignment from Leu to Thr. In Ashbya gossypii, secondary mutations enabled the new tRNA^His_UAG to be recognized by AlaRS instead of HisRS or ThrRS, therefore generating tRNA^Ala_UAG and reassigning CUN codons to Ala in this species. c ∣ Codon reassignment can affect bacteria and phage physiology. In a phage from the human oral cavity, reassigning UAG codons to Gln may allow the phage to interfere with translation of host genes without affecting translation of the phage genes. In the early stages of infection, the phage early genes contain few in-frame UAG codons and are therefore efficiently translated by the host machinery. Among these genes, the phage expresses release factor 2 (RF2), which suppresses translation of UGA codons, including recoded ones. As the bacterial protein RF1, which suppresses translation of UAG codons, contains multiple in-frame UGA codons, expression of RF2 by the phage inhibits translation of host RF1. This inhibition allows the phage to translate late-stage phage genes, which are enriched in recoded UAG codons, while modifying the translation of host genes. Part b is adapted from Su, D., Lieberman, A., Lang, B. F., Simonovic, M., Söll, D. & Ling, J., An unusual tRNA^Thr derived from tRNA^His reassigns in yeast mitochondria the CUN codons to threonine, Nucleic Acids Res., 2011, 39, 11, 4866–4874, by permission of Oxford University Press. Part c is adapted from Ivanova, N. N. et al. Stop codon reassignments in the wild. Science 344, 909–913 (2014). Reprinted with permission from AAAS.

Figure 4. Ambiguous decoding

a ∣ Ambiguous decoding refers to the process by which the same codon gives rise to incorporation of different amino acids in a nascent polypeptide chain. b ∣ Ambiguous decoding can result from errors in the aminoacylation reaction carried out by a specific aminoacyl-tRNA synthetase (aaRS), which loads a tRNA with a non-cognate amino acid; by multiple aaRSs recognizing the same tRNA, which leads to different amino acids being loaded onto tRNAs recognizing the same codon; or by ribosomal decoding errors. c ∣ In Candida albicans, altering the ratio of Ser to Leu incorporation at CUG codons introduces diverse cell and colony morphologies. Wild-type C. albicans encodes a CUG-decoding tRNA_CAG that is recognized by both seryl- and leucyl-tRNA synthetases. This ability of ambiguous decoding enables wild-type cells to display different colony morphologies, including smooth, ring, wrinkled and hyphae. Eliminating the ambiguity by the substitution of the tRNA_CAG with a Leu-specific tRNA results in loss of the smooth and ring morphologies. These changes also reduce cell adhesion and increase fungal susceptibility to macrophage killing by immune cells.

Figure 5. Expanding the genetic code with Sec and Pyl

a ∣ Protein synthesis with selenocysteine (Sec). Sec is biosynthesized on its tRNA. This occurs in multiple steps, beginning with Ser-tRNA^Sec formation catalysed by the normal seryl-tRNA synthetase (SerRS). In bacteria, Ser-tRNA^Sec is converted to Sec-tRNA^Sec by the Sec synthase (SelA). In archaea and eukaryotes, Ser-tRNA^Sec is first phosphorylated by phosphoseryl(Sep)-tRNA^Sec kinase (PSTK) to generate pSer-tRNA^Sec and then an enzyme related to SelA known as Sep-tRNA^Sec:Sec-tRNA^Sec synthase (SepSecS) converts pSer-tRNA^Sec species to Sec-tRNA^Sec. A specialized elongation factor (SelB) simultaneously binds to Sec-tRNA^Sec as well as the Sec insertion sequence (SECIS) element to direct recoding on the ribosome of specific UGA codons in selenoprotein mRNAs. b ∣ Protein synthesis with pyrrolysine (Pyl). Pyl is biosynthesized as a free amino acid in the cell. PylRS ligates Pyl to tRNA^Pyl, which contains an anticodon (5′-CUA-3′) that reads UAG codons. Like canonical aminoacyl-tRNAs, Pyl-tRNA^Pyl is bound by elongation factor Tu (EF-Tu), enabling UAG translation with Pyl on the ribosome. The Pyl system does not require a specialized elongation factor.