In the murky darkness, blue and green blobs are dancing. Sometimes they keep decorous distances from each other, but other times they go cheek to cheek — and when that happens, other colors flare.

The video, reported last year, is fuzzy and a few seconds long, but information technology wowed the scientists who saw it. For the first time, they were witnessing details of an early step — long unseen, merely cleverly inferred — in a central event in biology: the act of turning on a gene. Those blue and light-green blobs were ii fundamental bits of DNA called an enhancer and a promoter (labeled to fluoresce). When they touched, a gene powered up, as revealed by bursts of red.

Activation of a gene — transcription — is kicked off when proteins called transcription factors bind to two key bits of DNA, an enhancer and a promoter. These are far from each other, and no i knew how shut they had to come for transcription to happen. Here, working with wing cells, researchers labeled enhancers blue and promoters green and watched in existent fourth dimension. Also tweaked was the gene itself, such that mRNA copies, hot off the printing, would glow ruddy. The red flare is and so vivid it'south almost white, because several mRNAs at a time are being made. The study plant that the enhancer and the promoter have to practically bear on in order to kick off transcription.

CREDIT: H. CHEN AND T. GREGOR / PRINCETON UNIVERSITY

The consequence is all-of import. All the cells in our torso contain generally the same set of around 20,000 distinct genes, encoded in several billion edifice blocks (nucleotides) that string together in long strands of DNA. By awakening subsets of genes in different combinations and at different times, cells have on specialized identities and build startlingly dissimilar tissues: center, kidney, bone, encephalon. Yet until recently, researchers had no way of direct seeing only what happens during gene activation.

They've long known the wide outlines of the process, chosen transcription. Proteins aptly called transcription factors bind to a place in the gene — a promoter — as well as to a more distant Dna spot, an enhancer. Those two bindings allow an enzyme called RNA polymerase to glom onto the cistron and make a re-create of it.

That copy is processed a bit and then makes its fashion to the cytoplasm equally messenger RNA (mRNA). In that location, the cellular machinery uses the mRNA instructions to create proteins with specific jobs: catalyzing metabolic reactions, say, or sensing chemical signals from outside the cell.

This textbook take is true as far as it goes, but it raises many questions: What tells a given factor to plow on or off? How do transcription factors find the right sites to bind to? How does a gene know how much mRNA to brand? How do enhancers influence factor activity when they can be a million DNA edifice blocks away from the gene itself?

An infographic of a cell showing a close-up of the nucleus and outlining the steps and players involved in turning on a gene. The DNA helix forms a big loop, bringing together two distant regions of DNA, the enhancer and promoter. Various proteins, including transcription factors and mediator proteins, assist RNA polymerase as it makes an RNA copy of the gene. This copy, pre-mRNA, is processed into mRNA and then exits the nucleus via a pore. In the cell's cytoplasm, the mRNA is read, and the desired protein is synthesized.

For decades, scientists had only blunt and indirect tools to probe these questions. Ideas virtually DNA, RNA and proteins came from grinding up cells and separating components. Then, in the 1980s, scientists began using a game-changing technique called FISH (short for fluorescence in situ hybridization) to see DNA and RNA straight, right in the prison cell. Other methods followed — microscopes with improve resolution, new means to tag (and thus rails) players in this molecular symphony every bit it played out. Researchers could parse transcription every bit information technology happened, in detail.

Before, information technology was similar trying to hear the symphony by looking at a static picture of the orchestra, says Zhe Liu, a molecular biologist at the Howard Hughes Medical Institute'due south Janelia Research Campus in Virginia. "You would never effigy out what they are playing," he says. "Y'all could never appreciate how beautiful the symphony is."

Here'southward a gustation of what molecular biologists are learning by spying on this fundamental, nanoscopic process — increasingly in existent time, in living cells.

The life and times of transcription factors

Though scientists have long known that transcription factors dictate whether or not a gene powers up, information technology'due south been mysterious how these proteins navigate the ridiculously crowded space in the nucleus to find their binding sites.

Consider that, uncoiled, the DNA in a human being prison cell would run a meter or two long. The nucleus is virtually 5 to 10 micrometers in bore, so the packaging of our genome is alike to stuffing a string that could wrap 10 times around the Earth within a chicken egg, Liu says.

Researchers are simply starting to tackle how this coiling and looping affects gene transcription. For i thing, they suspect it could help explain how enhancers tin influence a gene'south activity from a great distance — because something far away when DNA is stretched out may be a lot closer when the genetic fabric is bundled up.

And if it seems miraculous that transcription factors know where they are going — well, near of them don't. By tracking these proteins in a unmarried jail cell over fourth dimension, researchers notice that they spend fully 97 percent of their life jiggling hither and thither, bouncing off of whatever $.25 of Dna they encounter until they luck out. (A few types may act equally leaders, scanning the genome, latching on to their target and setting up the correct conditions for a larger pack to follow.)

To come across how transcription factors move around inside the nucleus, researchers watched one specific transcription gene, Sox2, in living cells taken from mouse embryos. Shown are Sox2 molecules labeled with fluorescence, in a 3-D grid. Researchers recorded the movements of several Sox2 molecules within a single cell nucleus using a special microscopy approach that stacks 2-D images to make a 3D 1. Each of the traces represents the motion of a split up transcription factor.

CREDIT: J. CHEN ET AL / CELL 2014

I would imagine, at least, that when a transcription gene finally found its binding site, it could stay stuck and do its task for hours. Scientists used to believe so from experiments with dead, preserved cells.

But studies on live cells show that's far from truthful. Liu's lab and others have shown over the past v years that transcription factors bind but for seconds, and that high concentrations of them congregate near the bounden site, helping each other glom on. "Information technology'southward heed-boggling how transcription factors actually work," Liu says.

And there are a lot of them: Up to 10 pct of the genes in a mammal carry instructions for making ones of different flavors. Recent evidence suggests that this affords huge precision to the cell. For whatever given gene, varied combinations of transcription factors can ramp upwardly or tamp down the process, potentially making the system exquisitely tunable.

Central to all life is the transfer of information stored in our genes into molecules are used to brand proteins. How this happens has long been inferred but never directly seen. Now, scientists have captured videos of some of the key steps of factor expression – how a factor is "turned on" to brand a protein — in living cells. What they have constitute is surprising.

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Hooking up at the polymerase political party

If transcription factors are the gas pedal and brakes, the engine is RNA polymerase. In the bones model, RNA polymerase pulls apart a gene's two strands, then slithers down i of them to make an mRNA copy of it. Turns out things are a pilus more than complicated.

Studies in mashed-up and preserved cells had hinted that many polymerase molecules cluster together to make this mRNA magic happen. Just no ane had ever seen such a clump in living cells, and so no one knew how or when — or even if — the clumps formed. Past attaching a fluorescing chemical tag to RNA polymerase in alive cells, researchers saw multiple polymerases repeatedly group together for about five seconds — then scatter.

Last year, the same team of scientists spotted gatherings of other proteins as they congregated to assistance RNA polymerase do its task. These beasts — known as mediator proteins — form behemothic clusters numbering in the hundreds that join the RNA polymerases on the DNA.

Specialized groups of proteins called the mediator complex (light-green) assemble effectually a factor to help RNA polymerase practice its chore of copying DNA into mRNA (magenta). The box outline marks a iii-dimensional region surrounding the gene. The study showed that the two clusters fuse together and interact directly with the gene during transcription.

CREDIT: Westward. CHO ET AL / Science 2018

The 2 gaggles seem to concentrate into distinct aerosol, like blobs of oil in water. And then they fuse, mayhap creating a kind of self-assembling, cordoned-off transcription mill. A lesson from this? "Beyond the biochemistry, there are all these physical phenomena that may have a role in telling us how genes go turned on," says biophysicist Ibrahim Cissé of MIT, who led the work.

Messenger RNA is made in fits and starts

For decades, researchers assumed that when a gene is agile, transcription simply goes into "on" mode and cranks out mRNA at a steady clip. But a quantum technique called MS2 tagging, first developed in 1998 and however widely used, has radically changed that view.

Invented by prison cell biologist and microscopist Robert Singer and colleagues at the Albert Einstein Higher of Medicine in New York, MS2 tagging allowed scientists to see mRNAs in living cells for the very first time. (Key ingredients of the method come up from a virus called MS2 — hence the technology's name.)

In a nutshell, scientists employ engineering tricks so that mRNA fabricated from a specific gene bears distinctive structures called stem-loops. Through a 2d fob, those stem-loop locations are fabricated to glow fluorescently then researchers tin "see" mRNA from the gene of their selection whenever it is made and wherever it travels to, under a microscope and in existent time.

Vocaliser, who coauthored a 2018 article about mRNA imaging in the Almanac Review of Biophysics, used MS2 tagging to testify, with his colleagues, that the production rate of mRNAs from a gene fluctuates wildly over 25 minutes or and so. It turned out that the size of these bursts doesn't vary much, but their frequency does, and that's what dictates how energetically a gene pumps out its mRNA product. Increasing or decreasing the rate of this transcriptional "bursting" may let the system to ramp up or slow downward a gene's activity to encounter the jail cell'southward needs.

Researchers think that the on-off kinetics of transcription factors, meaning the charge per unit at which they pop on and off of their binding sites, somehow regulates transcriptional bursting. Merely they don't yet know how.

Trekking towards translation

Making mRNA is simply the offset stride in a gene's strutting its stuff. Next comes translating instructions in that mRNA to brand proteins. For that to happen, the mRNA must journey out of the nucleus and into the cytoplasm where the protein-making factories reside.

Scientists had causeless that the prison cell's molecular mechanism advisedly transported mRNA to the nucleus's membrane and then pumped it out into the cytoplasm. Using the same MS2 method, Singer'due south lab institute that wasn't and so. Instead, mRNAs bounce around — "buzzing around in the nucleus like a swarm of angry bees," as Singer terms it — until they happen to hitting a pore in the nuclear membrane. Only so does the prison cell'due south machinery lift a finger and actively shuttle mRNA through this gate.

In this video, proteins in the pores of the nuclear membrane are labeled cherry-red, and mRNA is labeled greenish. Using a special microscope designed to record at a very fast frame rate, researchers could watch private mRNAs every bit they zipped around the nucleus until they hit a pore and passed through the pore into the cytoplasm, where protein synthesis takes place.

CREDIT: D. GRÜNWALD AND R.H. SINGER / NATURE, 2010

More recently, Vocalizer and colleagues created mutant mice that enabled them to watch as mRNA shuttled up and downward a nerve cell's fragile dendrites, the structures that receive signals from other nerves. The team even got to spy on memory-making in action. The mRNAs they were tracking carried instructions for making a protein — β-actin — that is abundant in nervus cells and is idea to help bolster connections when memories are made in the brain. In a video that looks like a network of roads at nighttime, inside ten minutes after a nerve cell was activated, mRNAs cruised to points of contact with other fretfulness, ready for actin production to shore up those nerve-nerve connections.

Researchers devised a way to track mRNAs of a factor crucial for making memories as they traveled through living encephalon cells. The squad engineered a mouse so that all the mRNA copied from this cistron, which codes for a protein called β-actin, was labeled. β-actin helps neurons reshape tiny protrusions chosen spines that other neurons connect to, a process thought to be of import in learning and memory. When neurons grown in a dish were stimulated, β-actin mRNAs were produced in the nucleus within 10 to 15 minutes. In this video, you can run across about vi seconds of β-actin mRNAs cruising through the neuron'due south branches, or dendrites, after stimulation. The researchers believe that these mRNAs are searching the dendrites for spines that have only made connections, so that they tin synthesize β-actin protein right there on the spot.

CREDIT: HYE YOON PARK

Scads of details nearly factor activity remain mysterious withal, but it'south already clear that the process is far more dynamic than once assumed. "The change has been phenomenal, and it's accelerating rapidly," Singer says. "There's a lot of information to be gleaned simply by watching."