Enrichment of rare codons at 5' ends of genes is a spandrel caused by evolutionary sequence turnover and does not improve translation.

Previously, Tuller et al. found that the first 30-50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons. They argued that this slowed translation, and was adaptive because it queued ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined this 5' slow translation 'ramp.' We confirm that 5' regions are slightly enriched for rare codons; in addition, they are depleted for downstream Start codons (which are fast), with both effects contributing to slow 5' translation. However, we also find that the 5' (and 3') ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turnover relatively rapidly. When a new 5' end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5' ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5' end are expressed relatively poorly, and that substituting faster synonymous codons improves expression. Direct experiment shows that slow codons do not prevent downstream ribosome collisions. Further informatic studies suggest that for natural genes, slow 5' ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus, we conclude that slow 5' translation is a 'spandrel'--a non-adaptive consequence of something else, in this case, the turnover of 5' ends in evolution, and it does not improve translation.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

IMPORTANCE: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes ( rpoB, mmpL3, ndh ) and assessing their expression in the closely related and tractable model organism M. smegmatis . To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. IMPORTANCE: Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb . We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is the first report that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

Sirt4 Modulates Oxidative Metabolism and Sensitivity to Rapamycin Through Species-Dependent Phenotypes in Drosophila mtDNA Haplotypes.

The endosymbiotic theory proposes that eukaryotes evolved from the symbiotic relationship between anaerobic (host) and aerobic prokaryotes. Through iterative genetic transfers, the mitochondrial and nuclear genomes coevolved, establishing the mitochondria as the hub of oxidative metabolism. To study this coevolution, we disrupt mitochondrial-nuclear epistatic interactions by using strains that have mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from evolutionarily divergent species. We undertake a multifaceted approach generating introgressed Drosophila strains containing D. simulans mtDNA and D. melanogaster nDNA with Sirtuin 4 (Sirt4)-knockouts. Sirt4 is a nuclear-encoded enzyme that functions, exclusively within the mitochondria, as a master regulator of oxidative metabolism. We exposed flies to the drug rapamycin in order to eliminate TOR signaling, thereby compromising the cytoplasmic crosstalk between the mitochondria and nucleus. Our results indicate that D. simulans and D. melanogaster mtDNA haplotypes display opposite Sirt4-mediated phenotypes in the regulation of whole-fly oxygen consumption. Moreover, our data reflect that the deletion of Sirt4 rescued the metabolic response to rapamycin among the introgressed strains. We propose that Sirt4 is a suitable candidate for studying the properties of mitochondrial-nuclear epistasis in modulating mitochondrial metabolism.