Scientists Develop a Tool to Watch a Single Gene Being Transcribed in a Living Cell

Source: Memorial Sloan Kettering - On Cancer
Date: 07/05/2020
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A picture is worth a thousand words, or so the saying goes. But it can be quite a challenge to capture a picture of something that’s so tiny it’s on the scale of individual molecules.

The field of structural biology is dedicated to constructing images of very, very small things. But most of the techniques used by structural biologists to take these pictures require that the molecules are frozen in one position. This makes it difficult to watch the dynamic, shifting processes that are essential to life.

For the first time, researchers from the Sloan Kettering Institute have found a way to peer inside living cells and observe gene transcription. This is the process by which DNA is copied into messenger RNA (mRNA), which then specifies how a protein is made.

“Gene transcription is one of the most fundamental processes in all of biology,” says SKI structural biologist Alexandros Pertsinidis, senior author of the study, which was published in Cell. “We know that it’s highly regulated and uses complicated molecular machinery. Being able to watch this process as it happens is an important step forward in understanding what goes on inside cells.”

Following the Recipe

If you think of the genetic code as a universal cookbook, containing all of the instructions needed to make every part of a living organism, you can think of the various cell types as different restaurants, Dr. Pertsinidis explains. “French restaurants follow French recipes to make French dishes, and Italian restaurants follow Italian recipes to make Italian dishes,” he says. “In the same way, brain cells make the proteins that brain cells need to function, and liver cells make the proteins for liver function.”

A family of enzymes called RNA polymerases and a large set of factors that are associated with them regulate the transcription of individual genes and control the characteristics that cells exhibit. “The interplay between RNA polymerases and regulatory factors helps determine which genes are turned on and off in specific cells,” he says. “They also control how cells respond to outside signals, which can influence their activities.”

Until now, the function of RNA polymerases and associated regulatory factors has been studied indirectly, through biochemical reactions: Cells are broken open in a test tube and purified into individual parts. By adding or removing components and measuring the outcomes, scientists have been able to figure out certain molecular activities. How the machine as a whole works inside cells, however, has remained obscure.

“For 50 years, hundreds of researchers all over the world have studied these reactions,” Dr. Pertsinidis says. “But the problem has been that nobody has been able to directly observe how gene transcription happens inside a live cell.”

Zooming In on a Single Gene

In the study, the investigators used a highly specialized optical microscope to look at the activity of one RNA polymerase, called RNA polymerase II, as it interacted with genes and synthesized mRNA. The new method, developed by Dr. Pertsinidis’s lab, is called single-molecule nanoscopy.

To be able to look at the individual parts of cells, researchers label molecules with a fluorescent tag that makes them glow under the microscope. “But a cell is very crowded, and there are many reactions happening at the same time,” Dr. Pertsinidis says. “If you label all the polymerases in a cell, the whole nucleus is just a big glow.

“What’s new about this technology is the ultrasensitive, integrated system that lets us zoom in on a single tagged gene even when the cell nucleus is moving around and the specific chromosomal location is jiggling due to random microscopic motion,” he says. “At the same time, the system suppresses the signals from the other reactions that are happening, casting them into the background. This enables us to extract the signal for only the gene of interest and zoom in on it.”

The organization and dynamics of RNA polymerase II in the nucleus have been a topic of intense study over the past few decades. “Here, we directly observed the activity of this molecule and how it functions in the nucleus of live cells,” Dr. Pertsinidis says. “Being able to see how it interacts with other regulatory factors has unveiled the intricate hierarchies and interdependencies of these various factors. These insights enable us to reach a more detailed and comprehensive picture of transcription in live cells.”

Expanding to Other Cellular Processes

The researchers hope that their tool will be widely used to study complicated reactions inside living cells.

“There are enough details in our paper that other labs will be able to pick up and implement the technology,” Dr. Pertsinidis says. “We also have labs both inside and outside MSK that are interested in collaborating with us on specific projects.”

He adds that although he is focused on understanding gene transcription, the tools his team has developed could be used to study the details of other vital biological processes, such as DNA repair and protein synthesis.

Peering at Biological Molecules: A Conversation with Structural Biology Chair Christopher Lima

Source: Memorial Sloan Kettering - On Cancer
Date: 12/23/2019
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In June 2019, Christopher Lima was named Chair of the Sloan Kettering Institute’s Structural Biology Program. Dr. Lima replaced Nikola Pavletich, who had led the program since its creation in 2003 and who still has a lab in SKI. In addition to his new role, Dr. Lima is a Howard Hughes Medical Institute investigator and a faculty member in the Gerstner Sloan Kettering Graduate School of Biomedical Sciences.

We spoke with Dr. Lima about his research and his plans for the program. He also talked about the roles of technology and collaboration in advancing the field of structural biology.

What is structural biology?

Structural biology illuminates the function of important biological molecules through the study of their shape or architecture. What that means is being able to see at atomic or near-atomic resolution exactly how different molecules interact and activate or inhibit one another. It gives us a deep understanding of the mechanisms that are going on inside cells at the most basic level.

Inspiration on what to study can come from many areas. We may look at molecules that we know are essential for a fundamental process, like cell division. Or we may prioritize structural studies of gene products that we know are important because they are mutated in a disease or are targets of a drug.

What do you do in your lab?

We study pathways that are important for how RNA gets metabolized. There are many types of RNA, with some providing templates for translating genes into proteins. In addition, we study how proteins are modified after they get made, through a process called ubiquitination. We also use biochemistry to study these processes in test tubes.

We’ve been focused on these pathways for years because they are essential for life. Recently we’ve learned that several of the gene products that we work on may also be important for cancer. MSK-IMPACT is a test that looks for genetic changes in tumors. When that test was developed, DIS3ZCCHC8-ROS1, and XPO1 were among the genes identified as being mutated or altered in cancers. Having already studied these genes puts us in a better position to understand what they do and whether they might be a good target for intervention.

There was a lot of excitement when SKI got a cryo-electron microscope (cryoEM) about three years ago. How has that instrument changed structural biology research here?

X-ray crystallography, which is an alternative technique, requires us to crystallize molecules into a single shape or structure before we can study them. What’s great about cryoEM is that it allows us to study mixed samples that exist in multiple shapes or states at the same time. That’s extremely powerful. CryoEM also helps us study very large molecules or complexes that are sometimes difficult to crystallize.

At the same time, X-ray crystallography is still a valuable tool. It typically gives us higher resolution than cryoEM, albeit on smaller systems. This allows us to focus on the parts we’re particularly interested in studying in much greater detail.

Can you give an example of how the two technologies work together?

Let’s say we have an enzyme that’s important for a certain cellular function and we want to learn where a drug binds to it. CryoEM may be able to show us which part of the enzyme to focus on, but if we want to redesign the drug and make it more potent, we need to know every single point of contact between the drug and the enzyme in the active site. Depending on the problem, X-ray crystallography can often get to that level of resolution.

What’s unique about doing structural biology research at SKI?

It’s part of our mission to do innovative basic science. I would put SKI on par with some of the very best research universities in the world. We often collaborate with investigators in other parts of MSK as well as at other institutions.

SKI is populated by people who have a fundamental interest in discovering the systems and pathways that matter most in biology. If those pathways turn out to be relevant for disease, which is sometimes the case, it then opens up a world of possibilities for the translational and clinical research that’s also going on here.

What are your plans for the Structural Biology Program?

When I was a grad student, structural biologists had to pick a particular technique to focus on, whether that was X-ray crystallography or something else. Today, we take a hybrid approach to determining how biological pathways work. As such, we hope to recruit multidisciplinary scientists who define themselves not by the method they use but by the biological problem they’re focused on.

Twisting and Turning: Unraveling What Causes Asymmetry

Source: National Institute of General Medical Sciences - Biomedical Beat Blog
Date: 04/02/2020
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Asymmetry in our bodies plays an important role in how they work, affecting everything from function of internal systems to the placement and shape of organs. Take a look at your hands. They are mirror images of each other, but they’re not identical. No matter how you rotate them or flip them around, they will never be the same. This is an example of chirality, which is a particular type of asymmetry. Something is chiral if it can’t overlap on its mirror image.An image of a pair of hands, palms facing up. An arrow points to another image of the left hand on top of the right, both palms still facing up, illustrating that they can’t be superimposed. Our hands are chiral: They’re mirror images but aren’t identical.

Scientists are exploring the role of chirality and other types of asymmetry in early embryonic development. Understanding this relationship during normal development is important for figuring out how it sometimes goes wrong, leading to birth defects and other medical problems.

Decoding the Causes of Chirality

Michael Ostap , Ph.D., a professor of physiology at the Perelman School of Medicine at the University of Pennsylvania in Philadelphia, is studying how molecules interact to build cell structures that contribute to chirality in living things. His research focuses on the motors in cells, including a motor protein called myosin 1D, which plays an important role in generating chirality.

Dr. Ostap and his lab, along with Stéphane Noselli’s team  in France, examined how myosin 1D triggers chirality during the development of fruit flies. Dr. Noselli’s lab stimulated the production of myosin 1D during the early development of fruit fly organs that usually exhibit symmetry, including the epidermis (outer skin layer) and the trachea (similar to the windpipe). They found that the presence of this protein caused the cells to wind around each other in a spiral shape. The whole fly larva twisted into this spiral, and the spirals were chiral—they always turned in the same direction.

Understanding a Protein’s Push and Pull

Further examination by the Ostap lab revealed that myosin 1D induced spiraling of another protein called actin. Actin proteins form filaments required for cells to move and change shape. In this case, the researchers found that motor activity of the myosin changes the shapes of the cells, so they form tissues in a circular, counterclockwise geometry. How these molecular interactions lead to changes in cell shape, structure, and form remains a fascinating mystery, Dr. Ostap says. Unraveling these mysteries is an important step in developing better ways to treat certain diseases. 

The Ostap lab is continuing to study myosin 1D in flies with a bottom-up approach—from protein to cell to tissue. “We know that this protein is important for chirality,” Dr. Ostap explains. “We’re focused on the biophysical properties of why that’s the case. For example, we are studying myosin 1D’s biochemical and structural properties to try to learn more about how it makes these actin filaments turn.”

Dr. Ostap says studying myosin 1D’s activity in vertebrate research organisms such as mice, chickens, and zebrafish is important as well. A vertebrate’s body has many more components and more complicated interactions than a fruit fly’s body. But some of the same proteins may be important across organisms. “How similar these chiral cues are is not known yet known, and something we plan to study,” he says.

Dr. Ostap’s research is supported in part by NIGMS grant R37GM057247.

Atomic-scale view of bacterial proteins offers path to new tuberculosis drugs

Source: Rockefeller University, Newswire
Date: 02/03/17
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With the first detailed analysis of a cellular component from a close relative of the pathogen that causes tuberculosis, Rockefeller scientists are suggesting strategies for new drugs to curb this growing health problem. Each year, nearly half a million people around the world are infected with mutant TB strains capable of evading existing antibiotics.

The research, conducted by a Rockefeller team led by Elizabeth Campbell, in collaboration with scientists at Memorial Sloan Kettering, focuses on a cluster of interacting proteins called RNA polymerase. Crucial to all cells, this protein machine carries out a fundamental process in which genes within the DNA blueprint are copied into RNA. The RNA polymerase is the target of the antibiotic rifampicin—a lynchpin of modern TB treatment, which relies on a combination of drugs. Some bacteria become resistant to rifampicin by acquiring RNA polymerase mutations.

“Now that we can visualize the molecular machinery of the bacteria that the drug targets, we can use a structure-guided approach to better understand how the drug works, how bacteria become resistant to it, and how to potentially improve the drug’s action,” says Campbell, a senior research associate in Seth A. Darst’s Laboratory of Molecular Biophysics. She is one of the senior authors of a report published in the online journal eLife.

A molecular map

To visualize the structure the researchers used an imaging method known as x-ray crystallography. By crystallizing enzymes and other molecules interacting with each other—essentially freezing them in action—investigators are able to see how they fit together, much like keys fitting into locks. This ability to visualize what’s going on can point the way toward more effective drugs, which may be able to latch more securely onto enzymes and other molecules.

“Based on the findings reported in this study,” Campbell says, “we’re already investigating new compounds with new mechanisms of action that appear to inhibit the rifampicin-resistant version of TB. Our eventual goal is to get them into clinical trials investigating new treatments for TB, including rifampicin-resistant TB.”

Rockefeller chemist Sean F. Brady, who was not directly involved in the study, provided the team with these new compounds. He is now working together with Campbell, Darst, and other colleagues to further develop them into antibiotics and characterize the basis of their activity.

Not all bacteria are alike

It’s estimated that up to one-third of the world’s population is infected with M. tuberculosis, the bacterium that causes TB. In the study, the researchers worked with a closely related strain called M. smegmatis. “We needed hundreds of liters of cells to get enough of the material to do the crystallization,” says Elizabeth Hubin, a former Rockefeller graduate student who carried out much of the work. “M. tuberculosis grows too slowly to be able to collect the volume that’s needed, and it’s very dangerous to work with in the lab.”

But M. smegmatis relies on an RNA polymerase that is almost identical in sequence, structure, and behavior to the M. tuberculosis RNA polymerase, which led to another important finding in the study: The RNA polymerase from Escherichia coli, the bacterium most commonly used in lab research, is not. This means there may be a drawback to relying on E. coli as a model when developing certain types of antibiotics for bacteria that cause TB or other diseases.

“Most of the studies previously done with RNA polymerase were done using E. coli,” Campbell says. “We’ve always assumed that the enzyme works the same way in all bacteria, but our study shows we can’t assume what’s found in one bacteria applies to all bacteria.

“Every pathogen needs to be studied individually,” she adds, “so the field has a lot of work to do.”