New & Noteworthy

That Sweet Spot of Mistranslation

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.

Drunk man

Just as drinking too much alcohol can be toxic to people, too much mistranslation can be toxic to a cell. from pbs.twimg

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!

I can teach you lots of things about biology but you’ll need to learn about drinking alcohol on your own. Beer mug from Max Pixel (http://maxpixel.freegreatpicture.com/).

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

Tags: mistranslation , tRNA , MET22 , TTI2

Ticket to Transcribe

February 04, 2015


tRNACUG may not just be for translation anymore:

Just like a passenger needs a ticket to travel from one place to another, the Gln3 transcription factor needs a specific tRNA to travel from the cytoplasm to the nucleus. Image via Wikimedia Commons

Back before airplanes and cars, when times got tough people would often take trains to what they hoped were greener pastures.  And to hitch a ride on a train, they’d usually need to have a ticket. Turns out the same is true for Gln3, a transcription factor in yeast.

Basically, Gln3 stays in the cytoplasm as long as there are good sources of nitrogen available to the cell.  When these sources run out, Gln3 moves from the cytoplasm to the nucleus where it can turn on genes that can help the yeast cope with its new situation.

In a new study in GENETICS, Tate and coworkers have identified one of the tickets that lets Gln3 take the trip to the nucleus. And it was totally unexpected. To get to the nucleus, Gln3 needs a fully functional glutamine tRNACUG. No, really.

To get this evidence, Tate and coworkers used a reporter in which Gln3 was linked to GFP (green fluorescent protein). They tracked the location of Gln3 in the cell using fluorescence microscopy.

Using a temperature-sensitive mutant of tRNACUG, sup70-65, the authors showed that at the nonpermissive temperature of 30 degrees C, Gln3 could not translocate to the nucleus under a wide variety of conditions in which nitrogen was limiting. Gln3 had no problems translocating at the permissive temperature of 22 degrees C, and in wild-type cells Gln3 translocated at both temperatures. Clearly tRNACUG is doing something important in this process!

The next experiment showed that tRNACUG was more like a one-way ticket. Once Gln3 entered the nucleus under nitrogen starvation conditions at the permissive temperature, switching to the nonpermissive temperature had little effect. Gln3 stayed put.

A possible wrinkle in these experiments was that cells harboring sup70-65 formed chains reminiscent of pseudohyphae at the nonpermissive temperature no matter what the nitrogen conditions. One possible explanation for the results seen here was that many of these cells lacked nuclei. In this case, they might not see nuclear translocation because there was no nucleus to translocate to.

In the course of these studies, Tate and coworkers showed that adding rapamycin mimicked the effects of nitrogen starvation with one big difference—nuclear localization happened much more rapidly than with nitrogen starvation. This fast response allowed the authors to look at Gln3 localization while visualizing nuclei by staining DNA with DAPI (which gives a short-lived signal). They were able to use the DAPI to see that these cells did indeed have nuclei and that when they raised the temperature, Gln3 did not colocalize with the DAPI stained nuclei.  Gln3 was being kept out of nuclei at the nonpermissive temperature.

So it really looks like Gln3 needs a working tRNACUG to get into the nucleus. There are a couple of possible ways that this tRNA could be needed for Gln3 to make the trip.

In the first model, the tRNA is part of a complex that allows Gln3 to make the trip to the nucleus. In this model, it is almost as if Gln3 (or one of its compatriots) is clutching its ticket, tRNACUG. In the second, less fun model, the tRNA is required to translate a protein involved in Gln3’s transit. Which model is the correct one is still up in the air, but it will be interesting to see which is the right one.

This was the most astonishing finding in the article, but it was by no means the only one. We don’t have the time to go into the other experiments, which, among other things, teased apart differences in the four or five distinguishable pathways that work to turn on the cell’s nitrogen response.

This work highlights a recurring theme in basic research: we may think we know everything that’s going on (tRNAs just help to translate proteins, right?) but just about every time we look more closely, there is much more to see than first meets the eye. Being in the right place at the right time is essential, whether you’re escaping the Dust Bowl in The Grapes of Wrath or a transcription factor responding to the lack of a nutrient. It’s not so surprising that the cell has drafted every possible player into this process, even a lowly tRNA. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics


Since the title has this song stuck in our heads, we thought you might want to hear it too. Enjoy!

Categories: Research Spotlight

Tags: transcription factor localization , nitrogen utilization , Saccharomyces cerevisiae , tRNA

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