August 03, 2017
Anyone at a party knows that a little alcohol can make you charming, but a lot can doom any relationship from blossoming (just listen to Drunk Uncle!). In fact, too much can destroy a party! You need to have enough alcohol to curb social inhibitions, but not so much you overwhelm them.
According to a new study out in GENETICS by Berg and coworkers, something similar sometimes seems to be true when the genetic code evolves. A variety of beasts, including the yeast Candida albicans, have slightly different genetic codes—one or a few codons code for a different amino acid than usual. This is often the result of a mutated tRNA, the molecule that carries the right amino acid to the right codon.
These authors found that mutating only the anticodon, the part of the tRNA that recognizes the codon, is not a great way to head down the path to a new genetic code. That mutated tRNA leads to too many of one amino acid being replaced with another. Like the boorish drunk who kills the party because he has had too much to drink, the cell is overwhelmed with too many of the wrong amino acids scattered across its proteins and dies.
What Berg and coworkers found was that having fewer of these tRNAs with a mutated anticodon allowed for a tolerable level of amino acid substitutions. This means that this tRNA can hang around until it is helpful, like when it can suppress a new mutation of a key amino acid in a key protein.
The authors dubbed these low level mutated tRNAs as “phenotypically ambivalent intermediate tRNAs”—tRNAs that are in the process of changing the genetic code for at least one codon. It may be that the variants of the genetic code found in nature arose this way.
They started out with a strain of Saccharomyces cerevisiae with a mutation that inserted a proline in the wrong place of the TTI2 protein. This strain does extremely poorly in the presence of 5% ethanol.
The authors then tried to create and/or isolate suppressor mutations in a serine tRNA that could allow the strain to grow in the presence of 5% alcohol. The idea is that this tRNA would now carry a serine to that troublesome proline codon.
They started off by changing the anticodon of a serine tRNA to UGG. Now, the cell would put a serine in at CCA proline codons.
When they transformed a plasmid carrying this tRNA into their yeast strain, they got very few colonies. This obnoxious tRNA overwhelmed the cell by changing too many prolines to serines. It ruined the party!
They next set out to find a way to bring this bad boy under control. They mutated the serine tRNA with the proline anticodon using UV mutagenesis and found four mutants that allowed this yeast strain to grow in 5% ethanol.
Each mutant tRNA had a single mutation: G9A, A20bG, C40T, and G26A. Berg and coworkers set out to figure out why the cells now tolerated the mistranslation they couldn’t handle before.
What they found was that at least for two of them, G9A and G26A, the cells dealt better with the mutated serine tRNA because there was less of them around. The toxic drunk had become the tipsy charmer!
Well, maybe not quite charming, but at least something that could be dealt with. Both mutated tRNAs affected cell growth in the absence of ethanol, with the G26A version having the more severe effect—a reduction in growth by 70%.
Most likely there was less of the G26A variant because it was a victim of the rapid tRNA decay (RTD) pathway. The G26A variant affected the growth rate much less in the absence of alcohol in a strain deleted for MET22, a key gene in the RTD pathway.
By looking at the crystal structure of a serine tRNA in complex with its aminoacyl tRNA synthetase from Thermus thermophilus, Berg and coworkers predicted that the G9A mutation should result in a poorly folded tRNA. They found this was indeed the case when they compared the melting curves of the G9A mutant and the tRNA lacking the G9A mutation.
So what we have are some tolerable, but by no means benign mutations. For example, the G26A is quickly selected against in the absence of ethanol.
This makes it hard to imagine how these sorts of mutations might arise and one day permanently alter a genetic code. The key to understanding how this might happen is a set of experiments Berg and coworkers did that showed that both G26A and G9A have little or no effect on cell growth in the absence of a mutated anticodon. In other words, tRNAs can exist in a poised state, ready to easily adapt with a single change to the anticodon if need be.
And it turns out that poised tRNAs may exist in the real world. For example, human tRNAs have a lot of variation. Perhaps these are around to one day save a cell with a mutation that would normally be deadly.
As this work (and real life) shows, too much of a good thing can be bad. This is true of alcohol (remember high school or college?) and true of some mutant tRNAs. Yeast can teach us about the tRNAs, the rest we need to learn on our own. #APOYG
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight