In eubacteria, unlike eukaryotes, there is no evidence for selection favouring fail-safe 3’ additional stop codons

Abstract

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense ‘codons’ downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3’ sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates.

Readthrough errors purge deleterious cryptic sequences, facilitating the birth of coding sequences

“…We found that extensions with more ribohits tend to be shorter, implying selective pressure for shorter extensions in budding yeast. Previous studies have also shown evidence of selection for shorter extensions in eukaryotes (but not in bacteria; Ho and Hurst 2019). In yeast, backup stop codons are more frequent than expected by chance alone (Williams, et al 2004), show evidence of conservation (Liang, et al 2005), and are overrepresented in genes with a high codon adaptation index (Liang, et al 2005).…”

Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

“…This error rate can further be influenced by RNA secondary structures, or simply the immediate sequence environment of the stop codon, meaning that certain sequence constellations can lead to significant read-through rates on the order of 1–10% [ 34 , 48 – 50 ]. In eukaryotes, leaky stop codons appear to be generally selected against [ 51 , 52 ], although examples where read-through fulfills a regulatory function have been documented [ 53 , 54 ]. A fair amount of research has gone into exploiting leakiness therapeutically in cases where disease is caused by premature stop codons [ 55 , 56 ].…”

Variation in Release Factor Abundance Is Not Needed to Explain Trends in Bacterial Stop Codon Usage

“…Across bacterial species, the usage of the three stop codons varies considerably ( Povolotskaya et al 2012 ; Korkmaz et al 2014 ; Belinky et al 2018 ; Ho and Hurst 2019 ). This variation is not parsimoniously explained by simple covariation with GC content.…”

“…This variation is not parsimoniously explained by simple covariation with GC content. While TAA usage is negatively correlated with GC and TGA usage is strongly positively correlated, TAG usage, despite having an identical nucleotide content to TGA, is mostly low and unresponsive to GC pressure ( Povolotskaya et al 2012 ; Korkmaz et al 2014 ; Ho and Hurst 2019 ). In all bacterial species, on average about 20% of all stops is TAG no matter what their GC content (assayed as mean GC3 or GC of the whole genome).…”

“…It has additionally been postulated by Wei et al (2016) , by extension of the codon-anticodon adaptation hypothesis ( Ikemura 1981 , 1992 ; Akashi and Eyre-Walker 1998 ; Xia 1998 ; van Weringh et al 2011 ; Prabhakaran et al 2014 ; Chithambaram et al 2014a , 2014b ), that bacterial species adjust their stop codon usage to their relative expression of RF1 and RF2, a model we dub the “release factor (RF) hypothesis”. Not only would this explain the apparent preference for TAA, but also potentially the difference in the behavior of TGA and TAG usage against GC content ( Povolotskaya et al 2012 ; Korkmaz et al 2014 ; Wei et al 2016 ; Ho and Hurst 2019 ).…”