TOM JEFFERIESWhen recombinant protein expression fails, a scientist might blame faulty sequence data or a kit gone bad. But in the case of the ascomycete yeast Pachysolen tannophillus, researchers have identified a more fundamental problem: a CUG codon that normally translates to leucine instead results in alanine. This alternative coding, which joins another known nuclear sense codon reassignment in yeast, has been reported independently in two publications: a May Genome Research paper and another study, published this week (August 17) in PNAS. The authors of both papers have noted that their discoveries in P. tannophillus may be relevant to certain biotech applications involving the microbe as well as the study of RNA-based evolution in yeast.
The other known alternate sense codon use among yeast was first identified in Candida albicans two decades ago. This too involves a CUG codon, but results in serine in the translated protein.
“For this event to happen twice in a different linage is unexpected,” said Manuel Santos, the director of the Institute for Biomedicine at the University of Aveiro, Portugal, who was not involved with either study.
Santos, who has studied the CUG-serine reassignment extensively, told The Scientist that while a coding change may eventually confer an evolutionary fitness advantage, the initial reassignment comes at a substantial cost. Replacing bulky hydrophobic leucine with alanine or serine—both small, polar amino acids—could disrupt the structures and functions of critical proteins. Because the change is at the level of translation, researchers needed to compare sequences and codon usage across species to identify this hidden variation.
As part of a large phylogenetic study, researchers at the US Department of Energy’s Joint Genome Institute (JGI) in Walnut Creek, California, and their colleagues studied the alignment of predicted proteins in 700 orthologous genes from 29 different yeasts in an effort to identify conserved amino acids and determine which codons they came from. “It’s sort of a footprint of what the organism is using that codon for,” study coauthor Robert Riley, a bioinformatician at the JGI, told The Scientist.
For most species studied, CUG coded for conserved leucine between 70 percent and 86 percent of the time, but for P. tannophilus the standard coding occurred only 7 percent of the time, instead aligning 25 percent with alanine, the researchers reported.
To determine which amino acid was actually present in the proteins, Riley and colleagues extracted peptides from P. tannophilus grown in culture and analyzed them using liquid chromatography mass spectrometry (LC-MS). They found 178 identifiable peptides that mapped to coding sequences containing CUGs; 90 percent of them had LC-MS peaks indicating alanine and only 9 percent of the spectrum implied leucine. When they transformed P. tannophilus with either a wild-type or CUG-replaced, hygromycin–resistance gene, only the yeasts with the altered selection gene grew on antibiotic plates.
Codon modification may be required for researchers using yeast to express novel proteins in P. tannophilus and other yeast species, such as the workhorse Saccharomyces cerevisiae, Riley noted.
Beyond its potential effects on this and other biotech applications, CUG alanine coding might change the way scientists consider codon and transfer RNA (tRNA) evolution.
Martin Kollmar of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, who led the study published in Genome Research this spring, said he believes tRNA mutations drive the reassignment, based on his team’s structural analysis of 60 yeast species’ tRNAs. His group found that the P. tannophilus CUG tRNA’s secondary structure more closely resembles an alanine tRNA rather than either a leucine or serine one. The team’s LC tandem mass spectrometry (LC-MS/MS) data showed high levels of P. tannophilus CUG tRNA specificity, said Kollmar. He and his colleagues identified around 2,600 high quality peptides that matched coding sequence with CUG and confirmed that 97.2 percent translated the codon as alanine. The researchers observed a similar percentage when they performed the same analysis for the critical start codon, AUG, concluding that there is miniscule mischarging of alanine.
The potential causes and effects of alternative codon usage in yeast remain matters of debate. Meantime, there may be more forms of alternative coding to discover.
“With the genome sequencing efforts that are going on in fungi and other organisms, and the computation tools that we have to compare orthologous genes, [more coding variations] will be spotted,” said Santos.
S. Mühlhausen et al., “A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes,” Genome Research, doi:10.1101/gr.200931, 2016.
R. Riley et al., “Comparative genomics of biotechnologically important yeasts,” PNAS, doi:10.1073/pnas.1603941113, 2016.