New & Noteworthy

Sen1p Is the Traffic Cop for RNA Polymerase III

August 12, 2022

One way to imagine DNA is as a busy road with a lot of competing traffic. Say, a small village in southern Italy…where someone must mediate conflicts between competing vehicles to avoid disaster.

Illustration by Umberto Aiello, courtesy of the authors

It turns out that the “someone” in yeast cells is Sen1p. Two recent papers from associated groups describe the intriguing detail of how the Sen1p helicase plays this role for RNA polymerase III transcription. The paper by Aiello et al. in Molecular Cell shows how Sen1p regulates transcription-driven conflicts between the several machineries all engaged with DNA. In the related paper by Xie et al. in Science Advances, the authors show how the Sen1p helicase mediates “fail-safe” methods of transcription termination for RNA Pol III, thereby promoting efficiency and avoiding conflict with other pieces of machinery.

From Aiello et al., 2022

The key conflict preventing RNA Pol III from transcribing noncoding genes is with RNA Pol II, which is busy transcribing coding genes. Aiello et al. show how Sen1p has two strategies for mediating these conflicts, both of which involve interactions between Sen1p and the replisome. One involves temporary release of RNA Pol II from DNA while the other resolves genotoxic R-loops in nascent RNA. Both are critical for preventing genome instability.

from Xie et al., 2022

In the related paper by Xie et al., the authors focus on how termination of transcription of noncoding genes by RNA Pol III is achieved, and the role that Sen1p plays in termination. They show how Sen1p can interact with all three polymerases and also with the other two subunits (Nrd1p and Nab3p) of the NRD1 snoRNA termination (NNS) complex. More specifically, they show by mutation and co-immunoprecipitation that it is the N-terminal domain (NTD) of Sen1p that interacts with RNA Pol III and the replisome.

The authors use metagene analysis of RNA Pol II distribution at mRNA-coding genes to show how Sen1p can promote the release of RNA Pol II to resolve transcription-replication conflicts (TRCs). They further show how the association of Sen1p with the replisome is required for limiting TRCs at the ribosomal replication fork barrier, and how this action appears redundant with that of RNases H. The cooperation and redundancy in this role are key means to protect genome stability.

Not only is Sen1p required for termination of RNA Pol III transcription, but the authors show how this function is independent of the NNS complex. Unlike resolution of conflicts between RNA Pol II and RNA Pol III, the termination function of Sen1p does not require the replisome.

They asked the question of whether Sen1p acts via the primary termination site for RNA Pol III or, rather, a backup secondary termination that catches errors (i.e., when RNA Pol III reads through a weak termination site). Termination for RNA Pol III employs a tract of T nucleotides (T-tract) in the nontemplate strand and these T-tracts can be relatively weak or strong. When T-tracts prove insufficient to stop the polymerase, Sen1p plays a role by means of secondary structures in nascent RNAs, which act as auxiliary cis-acting elements. This backup method is termed the “fail-safe transcription termination pathway.” The RNA secondary structures are not absolutely required for RNAPIII termination, but can function as auxiliary elements that bypass weak or defective termination signals.

from Xie et al., 2022

Once more, it is the power of the yeast model that has allowed investigation to such exquisite molecular detail. That cells preserve genomic stability and avoid pile-ups amid so much traffic along DNA remains truly remarkable–even when we know more of how it works.

Categories: Research Spotlight

Tags: replisome, RNA polymerase II, RNA polymerase III, Saccharomyces cerevisiae, transcription, transcription conflicts

Mpe1p essential for mRNA processing

June 17, 2022

Messenger RNA (mRNA) 3′ end processing is an evolutionarily conserved and highly controlled process which requires several components from translation/transcription machinery. This processing involves monitoring nascent mRNAs for specific sequences, endonucleolytic cleavage, adding poly(A) tails, and triggering transcription termination. In budding yeast, the 3′ end processing machinery involves the cleavage and polyadenylation factor (CPF) complex and RNA-binding cleavage factors CF IA and CF IB. Based on the different enzymatic roles, the CPF complex has three distinct modules: polymerase, phosphatase, and nuclease.

CPF complex from Casañal A et al., 2017

A new study by Rodriguez-Molina et al. in Molecular Cell provides exciting insights into the role of the CPF complex in the 3′ end processing. The study provides strong evidence that Mpe1p, a nuclease module subunit of CPF, is involved in polyadenylation, cleavage, and transcription termination. The data show that in the presence of RNA, Mpe1p directly interacts with the polymerase module subunit Pfs2p through residues 207-268. The authors designate this region as a pre-mRNA-sensing region (PSR) of Mpe1p. However, another nuclease module subunit, Cft2p, hinders this Mpe1-polymerase module interaction. 

From Rodriguez-Molina JB et al., 2022

It is known that the polymerase module subunit Yth1p, interacts with RNA via a polyadenylation signal (PAS), a conserved sequence of A1A2U3A4A5A6. The authors show that in addition to Yth1p, Mpe1 interacts with PAS through the P215 residue. The authors hypothesize that Mpe1-PSR interacts with the polymerase module only after the Yth1p recognizes the PAS RNA, suggesting that Mpe1p may be able to ‘sense’ the RNA-polymerase module binding. 

Further, to understand the importance of the PSR region in Mpe1p, the authors analyzed mutants where its interaction with Pfs2p (mpe1-W257A,Y260A) or PAS (mpe1-P215G) is disrupted. Both mutants show reduced endonuclease and polyadenylation activities. Thus, suggesting that the same residues in Mpe1p involved in PSR-RNA/Pfs2 binding are also responsible in activating cleavage and regulating polyadenylation. A similar effect on endonuclease and polyadenylation activities is observed in a mutant where the CPF complex lacks the Mpe1p subunit, further corroborating the role of Mpe1p in mRNA 3′ end processing.

In the CPF complex, Mpe1p interacts with Ysh1p, another nuclease module subunit, via its N-terminal ubiquitin-like domain (UBL). This interaction stabilizes Mpe1p with the nuclease module (and CPF complex) even in the absence of RNA. To evaluate the importance of this interaction, a variant where the UBL region of Mpe1p is disrupted (mpe1-F9A,D45K,R76E,P78G) was generated. This variant is unable to form a stable CPF complex and show deficiencies in activating cleavage and polyadenylation activities. Thus the authors conclude that Mpe1p-Ysh1p interaction is essential for proper processing of 3′ end mRNA.

From Rodriguez-Molina JB et al., 2022

Another important aspect of mRNA 3′ end processing is the timely termination of transcription. The data show that the CPF complex lacking Mpe1p is unable to successfully terminate the transcription in time. Furthermore, the authors show that the PSR region specifically influences the role of Mpe1p in timely transcription termination.

Thus, the study highlights the role of Mpe1p as an essential subunit of the CPF complex in mRNA 3′ end processing, specifically in cleavage, polyadenylation, and transcription termination. 

Categories: Research Spotlight

Tags: CPF complex, Mpe1p, mRNA 3' end processing, Saccharomyces cerevisiae, transcription

New Twist on INO80 Chromatin Remodeling

June 03, 2022

The INO80 chromatin remodeling complex has long been the subject of intense study. Despite this, a recent report by Hsieh et al. in Molecular Cell reveals a new and unexpected biological activity: the INO80 complex (as compared to the other classes of chromatin remodelers) has a unique ability to act not only on nucleosomes but to enable transient detachment of an H2AH2B histone dimer to form smaller hexasomes, which are slid and repositioned differently from nucleosomes.

From Hsieh et al., 2022

Intriguingly, the authors demonstrate that the INO80 complex not only has the ability to act on hexasomes, but prefers to remodel hexasomes. Using in vitro biochemistry, they show that hexasomes are better substrates for the enzyme complex, better stimulate the enzyme’s ATPase activity, and are remodeled faster than full-size nucleosomes.

To explore the mechanisms underlying these observations, the authors asked about the acidic patches on H2A-H2B dimers. Given how previous studies had shown the importance of these patches for remodeling activity, the loss of one dimer of the two might be expected to hamper remodeling—not improve it. Instead, the team used a clever experiment with asymmetric nucleosomes containing mixtures of wild-type versus acidic patch mutant (APM) dimers to show how INO80 requires only a single acidic patch to maintain remodeling rates. 

From Hsieh et al., 2022

Arp5p is the protein within the INO80 complex that interacts most directly with acidic patches on histone H2 dimers. Using another series of in vitro experiments on reconstituted chromatin with a restriction enzyme accessibility assay and INO80(Δarp) (i.e. the complex lacking Arp5p), the authors show how the acidic patch specifically promotes formation of a key intermediate that primes the nucleosome for sliding along DNA.

From Hsieh et al., model of Arp5p binding the hexasome in a different conformation due to the absence of the dimer (right)

That these complex experiments are so informative relies on the long history of studying yeast genes and proteins. These newer studies build on the breadth of earlier examinations to look at the complex abilities of protein assemblies to perform both overlapping and unique biochemical actions. The study of how chromatin is opened to allow transcription in a regulated fashion remains a critical area of study, for which yeast is an ideal model.

Categories: Research Spotlight

Tags: chromatin, chromatin remodeling, hexasomes, INO80 complex, nucleosomes, Saccharomyces cerevisiae, transcription

New ncRNA Represses Zinc Regulator

February 04, 2022

In a twist to an established story, the termination of noncoding RNAs by the NNS complex (NRD1 snoRNA termination complex) appears to be dependent on the phosphorylation of a regulatory component. In the latest issue of Nucleic Acids Research, Haidara et al. show how the NNS-complex component Sen1p acts to repress transcription of the zinc master regular ZAP1 when Sen1p is phosphorylated, which appears to happen in response to excess zinc.

The NNS complex had previously been shown to terminate transcription of PHO84 via antisense RNA. In this current paper, the authors identify the previously unannotated noncoding RNA ZRN1 as lying directly upstream of ZAP1 and, when transcribed without termination, repressing the downstream gene. Termination of ZRN1 transcription by dephosphophorylated Sen1p derepresses ZAP1 mRNA levels via removal of the interfering RNA. As evidence of this relationship, a Sen1p phospho-mimetic mutation (T1623E) results in stable repression of ZAP1 transcription.

Working model for Sen1p regulation of gene expression, from Haidara et al.

The model proposed is that zinc excess leads to phosphorylation of Sen1p by an unidentified kinase, which then causes the level of ZRN1 transcript to increase because termination is impaired. This in turn represses ZAP1 mRNA levels by interference, thereby repressing genes responsible for increased zinc uptake and storage.  

Interestingly, the same system (i.e. Sen1p as a component of the NNS complex) represses PHO84 expression via interference, and PHO84 encodes a low-affinity Zn transporter that also contributes to zinc homeostasis. Might RNA interference play an expanded regulatory role over what is currently known?

Categories: Research Spotlight

Tags: noncoding RNA, Saccharomyces cerevisiae, transcription, zinc homeostasis

Move Along, Transcription Factor

December 07, 2016

Sumo wrestlers throw each other out of the ring. Just like sumoylation throws Gcn4p off DNA. Image from Wikimedia Commons.

For an election to go smoothly, people cannot stay too long in the voting booth. If a lot of people stayed in the booth and answered emails, sent texts, etc., after they finished voting, then the whole process would grind to a halt.

There is some evidence that activating genes may work similarly. The transcription factors (TFs) that bind DNA and turn up the expression of nearby genes can’t stay too long. If they do, the activation starts to peter out.

What is thought to happen with these sorts of TFs is that they bind their preferred DNA, and then once they have attracted the cellular machinery needed to read the gene, they are targeted for destruction. Then a new TF can bind and repeat the process.

In a new study in GENETICS, Akhter and Rosonina set out to investigate the process by which the yeast transcription activator Gcn4p is removed after it has bound DNA and done its job. Gcn4p activates a number of genes in response to amino acid starvation.

They found that a key step in the process is the addition of SUMO proteins to DNA-bound Gcn4p, which gets the ball rolling on the destruction of Gcn4p. Imagine a sumo wrestler settling in next to a voter once he enters the booth and then throwing him out if he tarries too long.

Their model is that once Gcn4p binds DNA, it is sumoylated. Then the DNA-bound, sumoylated GCN4 is further modified by kinases like Cdk8p, a component of the mediator complex which acts as a bridge between TFs and the cellular machinery responsible for reading a gene. This modified TF is then sent off to the 26S proteasome where it is degraded making room for an unmodified Gcn4p.

Previous research had shown that sumoylation of GCN4 required DNA binding. The first thing these authors did in this study was to determine if Gcn4p had to bind to its target DNA sequence in order to be sumoylated. It did not.

When they fused a mutant Gcn4p that could not bind DNA to the DNA binding domain of Gal4p, they found that this molecule was sumoylated at the correct places on the Gcn4p part of the fusion protein, lysines 50 and 58, when bound to a Gal4p binding site. Therefore, Gcn4p does not need to occupy its own DNA binding site in order to be sumoylated.

Another set of experiments showed that while DNA binding was required for sumoylation, interaction with RNA polymerase II (RNAP II), the enzyme that reads the genes that Gcn4p activates, does not appear to be necessary. For one of these experiments they used a temperature sensitive mutant of the largest subunit of RNAP II, Rpb1p, and showed that even at higher temperatures when RNAP II is inactive in these cells, DNA-bound Gcn4p is still sumoylated. In the other experiment they showed that DNA-bound
Gcn4p was still sumoylated when they used the “anchor away” technique to drag Rpb1p out of the nucleus and into the cytoplasm.

So DNA binding is sufficient, and the specific site is not important. And Gcn4p doesn’t have to be activated in order to be sumoylated.

Of course, turnover like this is a delicate thing. If Gcn4p is pulled off too soon, then it can’t activate as much as it might otherwise be able to do. This might affect the cell’s response to starvation just as much as Gcn4p staying put too long. Sort of like the sumo wrestler throwing a voter out of the voting booth before they could finish their voting can muck up the election.

Akhter and Rosonina created a fusion protein of Gcn4p and the yeast SUMO peptide Smt3p. Unlike Gcn4p, this protein is sumoylated before it binds DNA.

They found that yeast expressing this fusion protein fared less well under starvation conditions compared to yeast cells that expressed the wild type version of GCN4. And using chromatin immunoprecipitation (ChIP) analysis they showed that at least at the ARG1 gene, this was because there was less of the fusion protein bound under activating conditions.

So cells need for TFs to stay at the right place for the right amount of time. If they are pulled off too early or stay too long, the levels of activation can fall below what is best for the cells.

Unfortunately, we don’t have time to go over other experiments that tease out which kinases are important and when, but I urge you to read about them for yourselves. They take full advantage of the genetic tools available in yeast to make this sort of study possible…#APOYG!

Integrating all of this gives the following model:

Gcn4p model

Gcn4p is only a dimer when bound to DNA and this dimerization may be the signal for sumoylation by Ubc9p. A preinitiation complex forms through its interaction with the DNA-bound, sumoylated Gcn4p which brings in the enzyme RNAP II to transcribe the gene. Once the polymerase has left the nest, the kinase Cdk8p comes in and phosphorylates Gcn4p which signals Cdc4p/Cdc34p to ubiquitinate Gcn4p. The ubiquitinated Gcn4p is then degraded by the 26S proteasome opening the upstream activator sequence (UAS) up to a fresh, new Gcn4p.

Here, with the help of our super hero Saccharomyces cerevisiae, Akhter and Rosonina have dissected out what happens to a transcription factor once it binds to DNA (at least ones that bind for short times). It will be fascinating to see if this translates to other TFs in other beasts. While I love yeast for all it can do for us for bread, wine, beer, human health, helping solve world problems like climate change, and so on, I think my favorite use is still that it allows us to better understand the basic biology of how our cells work.

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

Categories: Research Spotlight

Tags: Cdk8, Gcn4, gene activation, sumoylation, transcription

Unleashing the Awesome Power of Yeast Transcription

October 07, 2015

With the right mutations, yeast can activate transcription over long distances—just as with the right social media, distance is meaningless for finding and organizing like-minded people. Image via

In the old days, before the internet, planes or even mass publishing, it was hard to spark a quick, worldwide movement. You simply couldn’t reach out to likeminded people who lived far away.

Nowadays things are very different. With the advent of social media, it is now trivially easy to spread the word. Using Twitter and Facebook, organizers can easily and effectively organize people who live on the other side of the world.

In terms of transcription activation, our friend Saccharomyces cerevisiae seems to be stuck in the old world. Its transcription factors can only turn up nearby genes. This is different from most other eukaryotic beasts, where activation at a distance is routine.

Except maybe yeast isn’t as backward as we think. It may be that yeast has the potential to activate transcription at a distance, but keeps that potential locked away.

This idea is supported in a new study out in GENETICS by Reavey and coworkers. These authors found that they could mutate away yeast’s inability to activate transcription at a distance.

In other words, this ability is there but is just prevented from being used. Yeast is keeping social media out of the hands of its genes.

Getting around social media/internet controls is not easy. Pressure might need to be applied at multiple points before people power is finally released through social media. And even when it does happen, it can sometimes be hard to figure out exactly why certain events tipped the balance.

Turns out that both of these are also true for long range transcription activation in yeast. Mutating a single gene was not enough—it took mutations in multiple genes to see any significant effect.

As you can imagine, it would be very tricky to hit all of the right mutations for a polygenic trait in one fell swoop. This is why Reavey and coworkers started their mutant hunt with a strain that could already weakly activate transcription from a distance, a strain in which the SIN4 gene was deleted. Now they just needed additional mutations to make the effect stronger.

In their screen they used a reporter in which the GAL4 upstream activating sequence (UAS) was placed 799 base pairs upstream of the HIS3 reporter. This reporter gives very low levels of activity in a wild type strain. They included a second reporter, the URA3 gene under control of the same upstream sequences, because Reavey and Winston had discovered in previous work using a single reporter that cis acting mutations and chromosomal rearrangements were a frequent source of false positive results.

The researchers put the reporter strain through multiple rounds of mutation with UV light and selection with increasing levels of 3AT, a competitive inhibitor of His3. After each round of selection, they measured mRNA levels for HIS3 and URA3 and chose strains that not only had higher 3AT resistance but also showed more transcription of the reporter genes.  In the end they found three strains that survived in the presence of galactose (to turn on the activator) and 10 mM 3AT.

As expected, each strain had multiple mutations. One strain had acquired mutations in the GRR1 and MOT3 genes. To confirm that these were the most important mutations, Reavey and colleagues engineered a fresh strain with just the original sin4 null mutation and the selected grr1-1 and mot3-1 mutations. The fresh strain completely recapitulated the selected strain, showing that these three mutations could unlock yeast’s potential for long-range transcriptional activation.

It makes sense that a grr1 mutation could affect transcriptional activation. Grr1 is a ubiquitin ligase that destabilizes Med3 (also known as Pdg1), a key component of the Mediator complex involved in transcription activation. The researchers provided evidence that this is how the grr1-1 mutation affects the process, by showing that mutating MED3 mimicked the effects of mutating GRR1.

It’s also not too hard to imagine how a mutation in Mot3, a sequence-specific transcriptional activator, could affect transcriptional activation, presumably by changing the expression of a gene under its control.

If you stay away from social media, you’ll lose opportunities for impromptu pillow fights. Who knows what yeast is missing without long-range transcription activation? Image via Wikimedia Commons

The results were not so clear-cut for two other strains that were selected. They arose from the same lineage, and each had acquired the ptr3-1 mutation on top of the original sin4 null mutation. One strain went on to further pick up the mit1-1 mutation, while the other got an msn2-1 mutation.

Again it isn’t too surprising that mutations in genes that encode sequence-specific transcriptional activators like Mit1 and Msn2 arose in these strains. But the selection of the ptr3 mutation in these lineages is something of a mystery.

It is hard to imagine how the usual job of Ptr3 in nutrient sensing and transport would be involved in keeping long range transcription activation down. Perhaps the researchers have uncovered a novel function for this gene.

And re-creating these two strains only partially restored the levels of transcription activation at a distance that were seen in the original strains. A little genetic detective work showed that a big reason for this was that both of the selected strains had acquired an extra copy of the chromosome that had the HIS3 reporter, chromosome III.

Reavey and colleagues deleted each of their identified genes to see which ones caused their effect through a loss of function mutation. Deleting GRR1, PTR3, and MSN2 all had the same effect as the original isolated mutations.

The same was not true for MOT3 and MIT1. Deleting either gene actually weakens long range transcription activation, suggesting that these two had their effect through gain of function mutations.

Finally, the researchers showed that the increase in long-distance transcriptional activation was not simply due to a general increase in transcription activation in the selected strains, by showing that their mutants did not have increased activity of a reporter with the GAL4 UAS placed 280 base pairs upstream of HIS3. In fact, if anything, the strains showed decreased activation with this reporter.

So this experimental strategy allowed Reavey and coworkers to identify some of the key genes involved in keeping transcription activation at a distance under control in yeast. In particular, they found compelling evidence that the Mediator complex is an important player. But there is still plenty of work to do. For example, which of the genes regulated by Mit1, Msn2, and Mot3 are important in long range activation? And what on Earth is Ptr3 doing in all of this?

The success of this approach also confirms that doing repeated rounds of selection in yeast is a viable way to select multiple mutants and study polygenic traits. This strategy may prove a boon for studying the many human diseases that are the result of polygenic traits.

Not only can we use yeast to uncover its activation potential, but we can also now potentially use it to uncover new treatments for human disease. Unleashing another awesome yeast power… 

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

Categories: Research Spotlight

Tags: polygenic trait, Saccharomyces cerevisiae, transcription

A Scientist Sees Transcription

September 23, 2015

While Horton uses his sensitive ears to hear a single Who, researchers need to use optical tweezers to see a single gene being transcribed. Image by Dave Parker via Flickr

In the classic Dr. Seuss tale Horton Hears a Who, the elephant Horton thinks he hears voices coming from a speck of dust. He gets into all sorts of trouble over this until all the Whos in Whoville prove they are alive when they all shout at once. Now Horton’s jungle compatriots believe him and Horton can hang out with his new friends.

Horton’s companions never get to hear an individual Who. They are not blessed with Horton’s big elephant ears and so have to just hear all the Whos shouting at once.

Up until recently, we have been in the same situation as the kangaroo and everyone else in the jungle when it comes to transcription in a cell. We can use all sorts of tools to get at what goes on when RNA polymerase II (pol II) gets ready and then starts to transcribe a gene, but we can only get an aggregate picture of lots of cells where it is happening. We can’t hear the Mayor of Whoville amidst all of the other Who voices.

In a new study out in Nature, Fazal and coworkers use the equivalent of elephant ears, optical tweezers, to study the initiation of transcription by purified pol II machinery from Saccharomyces cerevisiae on single molecules. And what they find is that at least for one part of the process, our having looked at things in the aggregate may have fooled us about how the process worked. It was important that we be able to pick out individual voices from the cacophony of the crowd.

Not surprisingly, transcribing a gene is tricky work. It is often split into three steps: initiation, elongation, and termination. And each of these can be subdivided further.

Fazal and coworkers focused on transcription initiation. Previous work had suggested that the process goes something like this:

Top image via Wikimedia Commons

Basically, an alphabet soup of general transcription factors and pol II sit down on a promoter. This complex then pries open the DNA and looks for a signal in the DNA to start transcribing. The polymerase then transcribes short transcripts until it shifts into high gear when it escapes the promoter and enters elongation phase.

This theory comes from the study of transcription in bulk. In other words, it derives from looking at many cells all at once or many promoter fragments in a test tube.

Fazal and coworkers set out to look at how well this all holds up when looking at single genes, one at a time. To do this they used a powerful technique called optical tweezers.

Optical tweezers can “see” what is going on with moving enzymes by measuring the change in force that happens when they move. For this study, the preinitiation complex bound to a longish (2.7 kb) piece of DNA was attached to one bead via pol II, the moving enzyme. The other end of the DNA was attached to a second bead. Each bead is then immobilized using lasers (how cool is that!) and the DNA is stretched between the two beads. Watch this video if you want more details on the technique.

Depending on where you attach the DNA to the bead, you can either track polymerase movement or changes in DNA by precisely measuring changes in the forces keeping the beads in place. Using this technique the researchers found that the bulk studies had done pretty well for most every step. Except for the initial transcribing complex.

The earlier studies had suggested that an open complex of around 15 nucleotides was maintained until elongation began. This study showed that in addition to the 15 base pairs, an additional 32 to 140 base pairs (mean of about 70 base pairs) was also opened before productive elongation could begin. And that this whole region was transcribed.

This result paints a very different picture of transcription initiation. Rather than maintaining a constant amount of open DNA, it looks like the DNA opens more and more until the open DNA collapses back down to the 12-14 base pair transcription bubble seen during elongation.

It turns out that this is consistent with some previous work done in both yeast and fruit flies. Using KMnO4, a probe for single stranded DNA, scientists had seen extended regions of open DNA around transcription start sites but had interpreted it as a collection of smaller, opened DNA. In other words, they thought they were seeing different polymerases at different positions along the DNA.

These new results suggest that they may have actually been seeing initial transcribing complexes poised to start processive elongation. Seeing just one complex at a time changed how we interpreted these results.

Fazal and coworkers were also able to see what happened to some of the 98% of preinitiation complexes that failed to get started. Around 20% of them did end up with an extensive region of open DNA of around 94 +/- 36 base pairs but these complexes were independent of transcription, as they didn’t require NTPs.

But since this opening did require dATP, they propose that it was due to the general transcription factor TFIIH, a helicase. It looks like in these failed complexes, TFIIH is opening the DNA without the polymerase being present.

A clearer picture of what might be going on at the promoter of genes starts to emerge from these studies. Once around 15 base pairs of DNA are pried open to form the appropriately named open complex, TFIIH unwinds an additional 70 or so base pairs. The polymerase comes along, transcribing this entire region. The whole 85 or so base pairs stays open during this process.

Eventually the polymerase breaks free and the opened DNA collapses back down to around 12-14 base pairs. Now the polymerase can merrily elongate to its heart’s content. Until of course something happens and it stops…but that is another story. 

Categories: Research Spotlight

Tags: optical tweezers, RNA polymerase II, Saccharomyces cerevisiae, transcription

Runaway Polymerases Can Wreak Havoc in Cells

October 16, 2014

A train without working brakes can cause a lot of destruction if it careens off the tracks. And it turns out that a runaway RNA polymerase II (pol II) can cause a lot of damage too.  But it doesn’t cause destruction, so much as disease.

Working brakes are important for both large and small machines, including RNA polymerase. Image from Wikimedia Commons

Unlike a train, which has its brakes built right in, pol II has to count on outside factors to stop it in its tracks. And one of these brakes in both humans and yeast is a helicase: Sen1 in yeast and Senataxin, the product of the SETX gene, in humans. 

Mutations in SETX are associated with two devastating neurological diseases: amyotrophic lateral sclerosis type 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2), both of which strike children and adolescents.  One idea is that these mutations may short circuit the brakes on pol II, causing it to keep on transcribing after it shouldn’t. And this is just what Chen and colleagues found in a new paper in GENETICS.

The researchers used the simple yet informative yeast model system to look at some of these mutations, and found that they disrupted the helicase function of Sen1 and caused abnormal read-through of some transcriptional terminators.  Looks like bad brakes may indeed have a role in causing these devastating diseases.

Some human proteins can function perfectly well in yeast. Unfortunately, Senataxin isn’t one of those; it could not rescue a sen1 null mutant yeast, so Chen and coworkers couldn’t study Senataxin function directly in yeast. But because Senataxin and Sen1 share significant homology,  they could instead study the yeast protein and make inferences about Senataxin from it.

First, they sliced and diced the SEN1 gene to see which regions were essential to its function. They found that the most important part, needed to keep yeast cells alive, was the helicase domain. But this wasn’t the only key region.

Some flanking residues on either side were also important, but either the N-terminal flanking region or the C-terminal flanking region was sufficient. Looking into those flanking regions more closely, the researchers found that each contained a nuclear localization sequence (NLS) that directed Sen1 into the nucleus. This makes perfect sense of course…the brakes need to go where the train is!  If we don’t put the brakes on the train, it won’t matter how well they work, the train still won’t stop.

These flanking sequences appeared to do more than direct the protein to the nuclear pol II, though.  When the authors tried to use an NLS derived from the SV40 virus instead, they found that it couldn’t completely replace the function of these flanking regions even though it did efficiently direct Sen1 to the nucleus.

Next the researchers set out to study the disease mutations found in patients affected with the neurological disease AOA2.  They re-created the equivalents of 13 AOA2-associated SETX mutations, all within the helicase domain, at the homologous codons of yeast SEN1.

Six of the 13 mutations completely destroyed the function of Sen1; yeast cells could not survive when carrying only the mutant gene. When these mutant proteins were expressed from a plasmid in otherwise wild-type cells, five of them had a dominant negative effect, interfering with transcription termination at a reporter gene. This lends support to the idea that Sen1 is important for transcription termination and that the disease mutations affected this function.

The remaining 7 of the 13 mutant genes could support life as the only copy of SEN1 in yeast. However, 5 of the mutant strains displayed heat-sensitive growth, and 4 of these showed increased transcriptional readthrough.

Taken together, these results show that the helicase domains of Senataxin and Sen1 are extremely important for their function. They also show that Sen1 can be used as a model to discover the effects of individual disease mutations in SETX, as long as those mutations are within regions that are homologous between the two proteins.

It still isn’t clear exactly how helicase activity can put the brakes on that RNA polymerase train, nor why runaway RNA polymerase can have such specific effects on the human nervous system. These questions need more investigation, and the yeast model system is now in place to help with that.

So, although it might not be obvious to the lay person (or politician) that brainless yeast cells could tell us anything about neurological diseases, in fact they can. Yeast may not have brains, but they definitely have RNA polymerase. And once we learn how the brakes work for pol II in yeast cells, we may have a clue how to repair them in humans.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: ALS, helicase, RNA polymerase II, Saccharomyces cerevisiae, transcription

Modifications? Heterochromatin Don’t Need No Stinking Modifications (for Activation)

May 01, 2014

When a character is asked to show his badge in the movie The Treasure of the Sierra Madre, he famously says something along the lines of, “Badges? We don’t need no stinkin’ badges!*” If histones in yeast heterochromatin could talk they might say something similar, except instead of badges they’d bring up modifications. Maybe something along the lines of, “Modifications? We don’t need no stinkin’ modifications for activation!” At least, they’d say this if a new study by Zhang and coworkers holds up.

The histones in yeast heterochromatin don’t need no stinkin’ modifications for gene activation. Image from Wikimedia Commons

In this study, the authors show that two different genes in the yeast S. cerevisiae are activated in heterochromatin in the absence of any significant changes to the surrounding chromatin.  This result is surprising because most researchers think activation and changes in chromatin always go hand in hand.  Apparently, in at least some situations they do not. 

This isn’t to say that chromatin didn’t do anything here…it most certainly did.  It served as a general damper on transcription.  But in this study chromatin was by no means the major player; it had a relatively small influence on the levels of basal and activated gene expression.  The authors suggest that this may be true for other genes in the more transcriptionally active euchromatin as well.

In the first set of experiments, Zhang and coworkers used a model system where the heat inducible gene HSP82 is flanked by the HMRE silencer from the HMR mating type cassette.  These silencers cause a 30-fold reduction in transcription of this hsp82-2001 transgene.   

Using chromatin immunoprecipitation (ChIP) the authors show that their transgene is indeed embedded in heterochromatin.  They see a lot of Sir3p around the promoter, a high density of histones that lack any of the telltale modifications of euchromatin, and very little RNA polymerase II (Pol II) or the mRNA capping enzyme Cet1p around the promoter.  These are all hallmarks of heterochromatin in yeast.

Things change when the yeast is subjected to heat shock.  Consistent with the observed 200-fold increase in transcription, they suddenly see lots of Pol II and Cet1p around.  But there is not a big change in the number of histones around the gene nor in their modifications.

When HSP82 is in its normal place in the genome, its activation is accompanied by specific acetylation and methylation of H3 and H4 histones.   In heterochromatin, despite significant induction, there is none of this.  The histones remain looking the same whether there is significant transcription or not. 

One trivial explanation for this might be that the chromatin is unaffected because the levels of transcription are lower than normal.  In other words, the lower final activity in the induced state is affecting histone modification. 

Zhang and coworkers rule this out by using a TATA-less HSP82 gene in euchromatin and show that all the appropriate histone modifications still happen.  This is true even though the damaged gene has 5-fold less activity compared with their transgene.  The low level of transcription does not appear to explain activation in the absence of histone modification.

Of course another reason for this unexpected observation might be that this pretty artificial construct isn’t representative of natural genes.  This doesn’t change the fact that its transcription is activated in the absence of histone modification, but it does question its relevance in the real world.

To address this issue, the authors looked for an inducible gene in natural heterochromatin and with a little bit of detective work, found the subtelomeric YFR057W gene.  No one knows what this gene does, but a close look showed a possible Stb5p binding site in its promoter. 

When Stb5p heterodimerizes with Pdr1p, the resulting dimer activates genes involved in pleiotropic drug resistance.  Indeed the authors found that YFR057w was induced 150-fold with a small amount of cycloheximide.  And when they used ChIP to compare the induced and uninduced states, they again found almost no changes in the chromatin around this gene despite an increase in the amount of Pol II and Cet1p.

Taken together these results suggest that activation doesn’t always have to come with chromosomal changes.  Which, while a bit surprising today, wouldn’t have turned any researchers’ heads a few decades ago.

In the old days (1980’s and 1990’s), a lot of focus was on how transcriptional activators might affect the ability of Pol II to load onto the DNA and to pry it open and start transcribing.  A lot of this was based on prokaryotic work where there really isn’t very much in the way of chromatin and a lot of activation depends on improving the ability of the polymerase to transcribe.

These days when people think about turning up a gene, they think about changing nearby chromatin.  Various enzymes work to modify histones at specific places, which both loosens up the chromatin to allow access by Pol II and serves as a way for various coactivators to recognize the DNA. 

As usual, reality is probably a combination of the two.  Activators can activate transcription in lots of different ways, some of which probably include chromatin changes while in others chromatin changes are simply a consequence of activation.  Not all transcription activation needs stinkin’ histone modifications.  

* This is actually a misquote that may have come from the Mel Brooks film Blazing Saddles.

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

Categories: Research Spotlight

Tags: heterochromatin, histone modification, Saccharomyces cerevisiae, transcription

Silly Sod’s Two Jobs

March 27, 2014

Most SGD users are probably too young to remember Saturday Night Live’s early years.  One very funny commercial parody involved Gilda Radner and Dan Aykroyd arguing over a product called Shimmer.  Gilda argues that it is a floor wax while Dan says it is a dessert topping.  In comes Chevy Chase to tell them that it is both.  Not quite as funny as Bassomatic, but still hilarious.

Not quite as weird as if this whipped cream were also a floor wax, but Sod1p being an enzyme AND a transcription factor was unexpected. Image from Wikimedia Commons

In a new study, Tsang and coworkers show something similar for the enzyme Sod1p.  Most people know Sod1p as an enzyme that protects the cell and its DNA by directly deactivating harmful reactive oxygen species (ROS) like superoxide.  Turns out that it may also be a transcription factor.

Now these two jobs aren’t quite as disconnected as a dessert topping and floor wax.  When Sod1p acts as a transcription factor, it is regulating genes that affect a cell’s response to ROS.  It is actually using its two functions to attack the same problem on multiple fronts.

Tsang and coworkers started out by looking at what happens to nuclear DNA under oxidative stress, using the Comet and TUNEL DNA damage assays. They found that endogenous and exogenous ROS caused DNA damage that was much worse in the sod1 null mutant – in other words, Sod1p protected the cells’ DNA. Using immunofluorescence, they also showed that Sod1p quickly went into the nucleus in the presence of ROS.  But if they restricted Sod1p to the cytoplasm by adding a nuclear export signal, the protein no longer protected the DNA.  In fact, it did no better than a strain deleted for SOD1.

In the course of these experiments one of the ways the researchers induced nuclear localization was with a burst of hydrogen peroxide.  But since hydrogen peroxide isn’t a substrate of the enzyme Sod1p, Tsang and coworkers next wanted to figure out how Sod1p got its signal to go nuclear.

Previous work had shown that SOD1 genetically interacted with MEC1, a yeast homolog of ATM kinases which sense oxidative stress.  They deleted MEC1 and found that Sod1p was trapped in the cytoplasm, unable to protect the cell’s DNA from damage.  This result was confirmed in human cells by showing that Sod1p only went nuclear if the cell made ATM kinase.

Tsang and coworkers suspected that this interaction might happen through a protein kinase called Dun1p, whose human homolog is a Mec effector. They confirmed a previous mass spectrometry result that showed Sod1p interacted physically with Dun1p.  And indeed, when DUN1 was deleted, Sod1p was again stranded in the cytoplasm.  Further work showed that Dun1p does its job by phosphorylating Sod1p on two serine residues, S60 and S99. When both these serines are mutated to alanine, preventing phosphorylation, less of the mutant Sod1p makes it into the nucleus. 

Using DNA microarrays, Tsang and coworkers next showed that SOD1 was required to activate 123 genes needed by the cell to respond to hydrogen peroxide.  These genes fell into five categories: oxidative stress, replication stress, DNA damage response, general stress response and Cu/Fe homeostasis.  The final experiment used chromosomal immunoprecipitation (ChIP) to show that in the presence of hydrogen peroxide more Sod1p was bound at the promoters of two of these genes, RNR3 and GRE2, but not the control gene ACT1

Of course, the authors have only looked at two of the 123 genes and an obvious next step is to figure out how many of the 123 have more Sod1p bound to their promoters in the presence of hydrogen peroxide.  Still, if these results can be confirmed and expanded they will suggest that Sod1p is able to combat oxidative damage in two completely different ways. 

In the first it uses its enzymatic activity to directly inactivate the ROS superoxide, while in the second it helps the cell respond to other ROS apparently by acting as a transcription factor.  While the jobs themselves are not as different as a floor wax and a dessert topping, how Sod1p goes about getting each job done is.  “Calm down you two, Sod1p is an enzyme AND a transcription factor.”

In addition to these two roles, we’ve written before about yet another regulatory role for Sod1p: it regulates glucose repression by binding to two kinases and stabilizing them. This is truly an overachiever of a protein!

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

Categories: Research Spotlight

Tags: oxidative stress, Saccharomyces cerevisiae, transcription