A facilitated diffusion confusion dissolution

To udirectbindfd1tilize the information contained within a cell’s genes, the enzyme RNA polymerase must find the beginning of each gene (the promoter).  Finding the beginning is a prodigious task:  RNAP must start at a particular base pair of DNA, but the cell contains millions of base pairs to choose from.  It has been proposed that gene-finding challenge is aided by a process termed ‘facilitated diffusion (FD).  In FD, RNA polymerase first binds to a random position on DNA and then slides along the DNA like a bead on a string until it encounters the target DNA sequence.

single-mol-testIn a recently published study in PNAS (1), biophysicists Larry Friedman and Jeffrey Mumm worked with Prof. Jeff Gelles in the Brandeis Biochemistry department to test key predictions of the FD model.  They used a novel light microscope that Friedman and colleagues invented and built at Brandeis, a microscope that can directly observe the binding of an individual RNA polymerase to a single DNA.  The scientists studied the σ54 RNA polymerase holoenzyme, an RNA polymerase found in most species of bacteria.  Surprisingly, none of the three predictions of the FD model that the experiments tested were found to be valid, demonstrating that target finding by the polymerase is not accelerated by sliding along DNA.  Friedman and colleagues instead propose that RNA polymerases are present in such large numbers that they can diffuse through the cell and efficiently bind to their target sites directly.  The absence of FD may explain how other proteins can bind to positions on the DNA that flank gene start sites and yet not interfere with RNA polymerase finding the gene.

Is this the end of the story? Not likely, given previous publications suggesting FD plays a role for some other DNA binding proteins. Using single-molecule techniques like those developed in the Gelles lab, scientists in next few years should give us a better idea if FD is very rare or very common. [editor: as a chemical engineer, I’m sad to see FD not have a role — it seemed like such a nice theory…]

Friedman LJ, Mumm JP, Gelles J. RNA polymerase approaches its promoter without long-range sliding along DNA.  Proc Natl Acad Sci U S A. 2013 May 29. [Epub ahead of print]



Dogic Lab Wins Andor Insight Award

The ‘Insight Awards‘  is a video contest showcasing research imagery from the physical and life sciences which utilize Andor technology to capture data.  This year, the Dogic Lab submitted a research video to the competition and garnered first prize in the Physical Sciences division for their video of Oscillating Microtubule Bundles.

From the competition notes:

Microtubules are a bio-polymer composed of the protein tubulin and are used extensively in the cell for cellular division, cell motility, and transportation of cargo within the cell. Here, we investigate the material properties of mixtures of microtubules, a depletion agent, and the molecular motor Kinesin. The microtubules, driven by Kinesin motors, spontaneously organize into bundles of microtubules that oscillate in a manner reminiscent of flagella and cilia found in biology. This engineered system will allow us to studying systems of self-propelled and self-organized matter that exist far from equilibrium in the field known as Active Matter.

We use standard fluorescent microscopy to image labeled microtubules in a thin, flow cell microscope chamber. An Andor Clara camera was used in conjunction with a Nikon Ti Eclipse microscope to capture this video.

Video and Entry by Stephen DeCamp.

For this, and more videos from the Dogic Lab, visit their YouTube page or their website at Brandeis University.

John Lowenstein (1926-2012)

Professor Gregory Petsko delivered the following tribute for John Lowenstein at Brandeis University Faculty Meeting late last year:

lowensteinJohn Lowenstein, who passed away from pancreatic cancer on November 3, 2012 at the age of 86, joined the Brandeis community in 1958 as a Senior Research Fellow, and became a member of the faculty two years later. From 1974-1995 he held the Helen Rubenstein Chair in Biochemistry; and he was also chair of the department in the 1990s.  John was not only a highly accomplished scientist; he was also an extraordinarily literate man, well versed in English, Russian and German literature, and a staunch devotee of opera, too. Even after his retirement in 2008, he continued to pursue his research interests and to work with students – he continued, in fact, to supervise an undergraduate until a few weeks before he died.

Those are the bare bones facts.  Let me tell you the story.  Many of you may know that John was one of the first members of the Biochemistry Department.  He came in the fall of 1958.  He’d been a Fellow at Oxford University, a position that allowed him a lot of independence, so when Mary Ellen Jones persuaded Nate Kaplan, the legendary founder of the department, to offer him a job, John already had the beginnings of a research program going.  He accepted the offer because America was, at that time, a better place for his wife, who was a clinician.  So he actually came here as an accompanying spouse (he once told me that he considered that a surprisingly liberated role for a man in the 1950s).  Before leaving England, he wrote to both the NIH and NSF to ask if it was OK for a foreigner to apply for a research grant; when they said it was, he wrote two different proposals, one to each of them.  He thought it was unethical to ask for salary on a grant, so he didn’t.  To his surprise, both grants were funded, so he ended up with full research support but no salary support.  Clearly, as we all know, Brandeis is a perfect choice for someone who wants to work without a salary!

Somebody then suggested he write a fellowship proposal, which he did, to the Helen Hay Whitney Foundation.  That was at the time, and still is, one of the most prestigious fellowships in the sciences.  Of course, he got that too, so when he finally showed up at Brandeis as, in essence, a postdoc, he had two grants and full salary support – more than most of the faculty!  Three weeks after he arrived, he started to lecture in Biochemistry on the 3rd floor of Kalman.  John once told me he was delighted that he was able to say that he had outlived that building!  Within 6 years of his arrivak, the Biochemistry Department was listed among the top 10 departments in the country, and it remained there until the mid-70s, when the practice of ranking departments stopped. 

After less than 2 years as a Whitney Fellow at Brandeis, John was already getting offers of faculty positions from other institutions for his work on nonenzymatic phosphate transfer by ATP, a very important process that he discovered.  Kaplan talked him out of considering most of them, but when one came from Tufts, Kaplan immediately promoted him to assistant professor.  By then, John had overcome his ethical objections to putting his salary on research grants…

purine nucleotide cycle

By the early 1970s, John had worked out the function of the important enzyme AMP-deaminase, the founding member of a family of enzymes that are very important in health and disease.  He then went on to do something only a handful of scientists have ever done: he discovered a metabolic pathway, the purine nucleotide cycle that AMP deaminase functions in.  This ought to be called the Lowenstein Cycle, but John once told me that if you discover something so important that you don’t have to name it after yourself, you’ve really done something special!

John always ran a small research lab but in many ways he ran the department for quite some time.  He had served on every possible departmental committee, popular and unpopular, sometimes all at the same time, or at least it seemed that way to him!  He was Chair of the Department in the early 1990s.  When I became chair of the department, a few years ago, I immediately sought out his advice.  He said, “Greg, my advice to you is to start drinking heavily.” 

He taught Biochem 101, the department’s signature graduate course, for many years, and then led the movement for the department to teach undergraduates.  Putting his money where his mouth was, he then taught the basic undergraduate course, Biochem 100 – sometimes two sections a day – until 2005. 

John had three sons; his middle son is a scientist at Johns Hopkins; the youngest is a professor of music, and his oldest is a businessman.  John was very, very proud of his family, but said to me on more than one occasion that the major place in his life, outside that family, was Brandeis.  It’s no accident that, for many years, John was the faculty member all the graduate students went to for advice.  He had a pilot’s license and used to fly sailplanes, but I think the students were quick to identify someone who was always good at keeping his feet on the ground. 

John once said that if he were independently wealthy he would still do what he does.  I was thrilled to hear that because it meant that, even after becoming emeritus, John would still be around a lot, and he was.  The best raconteur in the department, John had a warm, wise and often dryly funny story for every occasion.  It was part of the way he imparted his enormous common sense.  No one here meant more to me as a colleague, a friend, and a role model. He said to me that, when he retired, it meant the Biochemistry Department was going to gain in reputation, because he was going to have much more time for research… Few did it better, or with more style. 

On the occasion of his retirement, I asked John to sum up his years at Brandeis.  He just smiled and said, in his typical understated way, “I like to think I’ve been a cog in something worthwhile.”  We should all be such a cog!

Remembrances may be made to the American Jewish Joint Distribution Committee, www.jdc.org.

Pieter Wensink (1941-2012)

Professor Jim Haber presented the following memorial tribute at Faculty Meeting on Nov 8, 2012:

Professor Emeritus Pieter Croissant Wensink passed away on October 2, 2012 in Wellesley, MA. Pieter was born in Washington, DC, in 1941, and grew up in Bethesda and Chevy Chase, MD. He attended Lawrence College in Appleton, WI, but like many young people in the 60s, dropped out. He ended up working in a laboratory at Johns Hopkins, where he discovered a passion for science. He never got his BA, but by taking night courses Pieter got himself accepted as a graduate student at Johns Hopkins, where he received his PhD in Biology in 1971, working with Don Brown, a pioneer in studying the regulation of gene expression in frogs. Pieter then went to Stanford, where he did post-doctoral work with David Hogness. At Stanford, Pieter got in on the ground floor of the new recombinant DNA technology. He published, with Hogness, a landmark paper entitled “A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster” – the fruit fly.

In 1975 Pieter came to Brandeis as an Assistant Professor in the Rosenstiel Center and in the Department of Biochemistry, bringing to Boston the then-rare and prized knowledge of how to clone genes. I remember clearly in 1976 when an MIT professor, David Botstein, and his postdoc, Tom Petes, camped out at Brandeis for several weeks learning from Pieter how to clone yeast genes. Their collaboration resulted in another major paper “Isolation and analysis of recombinant DNA molecules containing yeast DNA.” Soon thereafter Matthew Mesleson arrived from Harvard, to collaborate with Pieter on the “Sequence organization and transcription at two heat-shock loci in Drosophila.” All of these papers were pioneering works.

Pieter also taught these “dark arts” to the people in my lab and launched us and others at Brandeis on the way to understanding the mysteries of chromosome architecture and gene regulation. In 1981 Pieter also wrote a book in collaboration with his Biochemistry colleague Bob Schleif: Practical Methods in Molecular Biology.

Pieter’s own work, carried out with a series of superb graduate students, focused on genes that encode the proteins that make up the yolk of Drosophila eggs. The study of these genes revealed the complicated way that yolk protein genes are turned on only in females and only in their ovaries. Many of Pieter’s students are now Professors in their own right at major universities around the country.

In the early 1990s Pieter was diagnosed with a benign brain tumor – a meningioma – that required two surgeries to extirpate. Probably his tumor was the result of the now-impossible-to-believe treatment of a ringworm infection with X-rays when he was about 2 years old. The second operation left him unable to concentrate as he had, and Pieter, sadly, decided that he could no longer run his lab or give the clear lectures had had been offering. So he left Brandeis as an emeritus Professor with a medical disability. Pieter was remarkably calm and accepting about his situation. He decided to pursue a long-deferred passion to paint, and some years ago he earned his BFA with distinction in painting from the Massachuetts College of Art. Altogether, Pieter had 5 operations on the cancers that led to his death.

Pieter’s greatest joy in life was his family. He was married to Dorothy E. (Perry) for 43 years and was the devoted father of Tom, Alan and Joe (who recently earned his PhD in English from Brandeis).

Most of you never met Pieter, so I thought it would be good to see Pieter and some of his colleagues as we looked in the late1970s (Pieter, Michael Rosbash, Marion Nestle (now oft-interviewed nutritionist at NYU), myself, and David DeRosier). And to see two of his paintings. He was a fine man.


Deciding the fate of a stalled RNA polymerase

Ever wondered what happens when the transcription machinery runs into a DNA lesion or a protein roadblock? Alexandra M. Deaconescu, corresponding author and research associate in the Grigorieff laboratory together with HHMI Investigator and Biochemistry Professor Dr. Nikolaus Grigorieff and Dr. Irina Artsimovitch (Ohio State University) address this question in a new review “Interplay of DNA repair with transcription: from structures to mechanisms” featured in the latest issue of Trends in Biochemical Sciences. The review describes emerging mechanisms of transcription-coupled DNA repair with emphasis on the bacterial system.

Materials in Motion: Engineering Bio-Inspired Motile Matter

Life is on the move! Motion is ubiquitous in biology. From the gargantuan steps of an elephant to the tiniest single celled amoeba, movement in biology is a complex phenomenon that originates at the cellular level and involves the organization and regulation of thousands of proteins. These proteins do everything from mixing the cytoplasm to driving cell motility and cell division. Deciphering the origins of motion is no easy feat and scientists have been studying such complex behavior for quite some time. With biology as an inspiration, studying these complex behaviors provides insight into engineering principals which will allow researchers to develop an entirely new category of far-from-equilibrium materials that spontaneously move, flow or swim.

In a recent report in the journal Nature, a team of researchers from Brandeis University consisting of Tim Sanchez, Daniel T. N. Chen, Stephen J. DeCamp, Michael Heymann, and Zvonimir Dogic have constructed a minimal experimental system for studying far-from-equilibrium materials. This system demonstrates the assembly of a simple mixture of proteins that results in a hierarchy of phenomena. This hierarchy begins with extending bundles of bio-filaments, produces networks that mix themselves, and finally culminates in active liquid crystals that impart self-motility to large emulsion droplets.

Their system consists of three basic components: 1) microtubule filaments, 2) kinesin motor proteins which exert forces between microtubule filaments, and 3) a depletion agent which bundles microtubule filaments together. When put together under well-defined conditions, these components form bundled active networks (BANs) that exhibit large-scale spontaneous motion driven by internally generated active stresses. These motions, in turn, drive coherent fluid flows. These features bear a striking resemblance to a biological process called cytoplasmic streaming, in which the cellular cytoskeleton spontaneously mixes its content. Additionally, the system has great potential for testing active matter theories because the researchers can precisely tune the relevant system parameters, such as ATP and protein concentration.


The researchers also demonstrate the utility of this biologically-inspired synthetic system by studying materials science topics that have no direct biological analog. Under dense confinement to an oil-water interface, microtubule bundles undergo a spontaneous transition to an aligned state. Soft matter physics describes such materials as liquid crystals, which are the materials used to make liquid crystal displays (LCDs). These active liquid crystals show a rich variety of dynamical behavior that is totally inaccessible to their equilibrium analogs and opens an avenue for studying an entirely new class of materials with highly desirable properties.

Lastly, inspired by streaming flows that occur in cells, the researchers encapsulate the bundled active networks into spherical emulsion droplets. Within the droplet, microtubules again formed a self-organized nematic liquid crystal at the oil-water interface. When the droplets were partially squished between glass plates, the streaming flows generated by the dynamic liquid crystals lead to the emergence of spontaneous self-motility.

This research constitutes several important advances in the studies of the cytoskeleton, non-equilibrium statistical mechanics, soft-condensed matter, active matter, and the hydrodynamics of fluid mixing. The researchers have demonstrated the use of biological materials to produce biomimetic functions ranging from self-motility to spontaneous fluid flows using fundamentally new mechanisms. Additionally, the experimental system of bundled active microtubules is poised to be a model for exploring the physics of gels, liquid crystals, and emulsions under far-from-equilibrium conditions.

To see more videos from the Dogic lab at Brandeis University, check out their YouTube page.

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