New & Noteworthy
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October 29, 2012
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When Polymerases Collide
October 25, 2012
Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.
There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.
What happens when RNA polymerases meet head-on?
Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!
Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.
As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.
For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.
When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.
They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.
Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.
Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?
To get past the Black Knight, Arthur had to destroy him. Hopefully the cell has more tricks up its sleeve than that!
One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.
The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.
Tags: RNA polymerase II > Saccharomyces cerevisiae > transcription > ubiquitin-mediated degradation
Better Beer Through Beards
October 16, 2012
Small time craft brewers are always looking for ways to push the envelope of beer taste. They are trying to find variations in beer’s fundamental ingredients — hops, barley, and yeast — that will make their beer distinctive. Of these three, the most important is probably yeast (of course, we’re biased here at SGD!).
Think of the tasty beers that could come out of those beards.
Something like 40-70% of beer taste comes from the yeast used to make it alcoholic. This is why brewers search high and low for new strains of yeast that will give their beer that special something which will make it stand out. They have looked on Delaware peaches, ancient twigs trapped in amber, Egyptian date palms, and in lots and lots of other places.
But brewers don’t always have to go far away because sometimes the best yeast is right under their noses. Literally.
A brewery in Oregon found the yeast they were looking for in one of their master brewers’ beards. They are now using this yeast to brew a new beer! This seems uniquely revolting but the beer supposedly is quite tasty. Perhaps if they don’t advertise the source of their yeast, this beer could become popular.
They aren’t sure where the yeast in his beard came from, but they think it may have come from some dessert he ate in the last 25 years or so (he hasn’t shaved his beard since 1978). What would be fun is if his beard wasn’t just an incubator, but a breeding ground for new yeast. Maybe yeast from a dessert from 1982 hooked up with a beer yeast blown into his beard while he was working at the brewery. The end result is a new improved hybrid yeast!
Of course we won’t have any real idea about this yeast until we get some sequence data from it. And all kidding aside, the more yeast that are found that are good for making beer, the better the chances that scientists can home in on what attributes make them beer worthy. So this beard borne yeast may help many beers in the years to come despite its troubling beginning.
Perhaps brewers also need to start searching through more beards to look for likely beer yeast candidates. Beard microbiome project anyone?
More information
Original lager yeast found in Patagonia
SGD Hardware Update
October 9, 2012
We are in the process of migrating SGD servers to new faster hardware! You may have already noticed an increase in performance. There could be some teething issues in the next couple days – so please bear with us!
How To Remember Without a Brain
October 2, 2012
Single celled beasts like the yeast S. cerevisiae can “remember” previous insults and so respond better to environmental changes in the future. For example, a yeast cell treated with 0.7M NaCl will respond better in the future to hydrogen peroxide. Not only that but so will its daughters, granddaughters and even its great, great granddaughters.
Who needs a brain to remember things?
In a new study out in GENETICS, Guan and coworkers show at least a couple of ways that this can happen. One is what anyone in biology might expect these days (although with an interesting twist). The NaCl treatment causes a rewiring of the regulatory network at an epigenetic level and this affects future responses to environmental insults.
But fancy epigenetic changes aren’t the only way that yeast remembers things. No, it also uses a simple, elegant solution—protein stability.
Long Live The Protein!
The researchers did a set of experiments that showed that a yeast’s memory of a salt treatment did not rely on new protein synthesis and that it slowly faded with each generation. One possible explanation was that the salt induced a stable factor that was divvied up and diluted with each passing generation. Guan and coworkers found that this was the case and that at least one of these factors was the cytosolic catalase 1 protein, Ctt1p.
The cytosolic catalase 1 or CTT1 gene is induced by salt but quickly returns to normal levels when the salt is removed. However, Ctt1p is so long lived that it hangs around for at least six hours. In that time the yeast has budded off multiple daughters, all of which are still better at dealing with hydrogen peroxide than their untreated sisters.
What a marvelously simple way to adapt! Just make something that hangs around a long time and you and your kids will do better when the next insult comes. The elegance of evolution.
This explains in part how yeast cells can remember the salt treatment of their ancestors, but a single long-lived protein isn’t the whole story. No, there is something a bit more complicated going on at the nuclear pore too.
Attached for Quick Access
Guan and coworkers looked at the gene expression pattern of salt stressed and naïve yeast when exposed to hydrogen peroxide. They found that 449 genes responded more quickly to hydrogen peroxide treatment if the cell had been pretreated with salt. Importantly, 51 of these hadn’t reacted previously to the salt treatment, meaning that previous activation wasn’t required.
One idea is that these genes are more accessible to transcription because they are associated with the nuclear pore. The idea is that faster response happens because the gene is closer to the nuclear envelope and/or because it has been looped near some sort of activator.
This is what has been proposed with inositol starvation and it looks like it may be true here too. In both cases, eliminating Nup42p, a nuclear pore protein, eliminates the more rapid response to hydrogen peroxide.
So in this case it looks like cells can remember a previous insult with just a long-lived protein and a bit of genetic rewiring. It will be interesting to see how universal these sorts of mechanisms are for cell memory.
Tags: catalase > cellular memory > Saccharomyces cerevisiae > stress response
