September 23, 2015
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