“Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing”

Mcm2-7 is a ring-shaped DNA helicase that plays an essential role in DNA repliction in eukaryotic cells.  Two of the helicase molecules must encircle the double-stranded DNA at a replication origin, establishing a loaded, anti-parallel double-ring complex able to start replication at the appropriate cell cycle stage.  In this study, Simina Ticau together with collaborators from Steve Bell’s lab (MIT), Jeff Gelles’ lab (Brandeis), and New England BioLabs used wild-type and mutant helicases in single-molecule colocalization (“CoSMoS”) and single-molecule fluorescence resonance energy transfer (smFRET) experiments to identify the mechanisms by which regulatory factors and nucleotide hydrolysis control ring opening and coordinate loading. This work reveals the molecular processes that serve to prevent catastrophic genome damage due to incorrect or mistimed assembly of the replicative machinery.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing
Simina Ticau, Larry J Friedman, Kanokwan Champasa, Ivan R Corrêa Jr, Jeff Gelles, Stephen P Bell
Nat. Struct. Molec. Biol. (2017) 24: 309–315.

Video: Radcliffe Institute for Advanced Studies lecture

In February, Jeff gave a public lecture on “Seeing the Birth of an RNA Molecule” at the Radcliffe Institute for Advanced Studies.  This talk, intended for a scholarly audience consisting of both scientists and non-scientists, used single-molecule studies of transcription as examples of how visualization of molecular behavior has led to new insight into the mechanisms of fundamental molecular processes in biology.

Dynamics of GreB-RNA polymerase interaction

In living cells, messenger RNAs are not manufactured by RNA polymerases (RNAPs) functioning alone.  Instead, RNA synthesis is carried out collectively by RNAP together with accessory proteins that associate with the RNAP-containing transcription elongation complex and modulate its activity.  In this paper, Larry Tetone, Larry Friedman, and Melissa Osborne, along with their collaborators from the Gelles and Landick labs, used multi-wavelength single-molecule fluorescence methods to for the first time directly observe the dynamic binding and dissociation of an accessory protein with an RNAP during active transcript elongation.  The protein, GreB, is important for transcript proofreading in E. coli and other bacteria and is a functional analog of the TFIIS protein in eaukaryotes.  “Unexpectedly,” the authors report, “GreB was not selectively recruited to RNAPs requiring its transcript proofreading function. Instead, GreB transiently bound to multiple types of complexes, eventually finding via random search RNAPs that require its activity. The observations suggest a paradigm by which a regulator can act while minimizing obstruction of a binding site that must be shared with other proteins.”

Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue
Larry E. Tetone, Larry J. Friedman, Melisa L. Osborne, Harini Ravi, Scotty Kyzer, Sarah K. Stumper, Rachel A. Mooney, Robert Landick, and Jeff Gelles
PNAS (2017) 114:E1081-E1090.

“The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers”

The physical forces that drive oligomerization of soluble proteins are well understood and have been extensively studied.  For proteins with transmembrane domains — transport enzymes, for example — oligomerization is often essential for function but its physical basis is less clear.  In this project,  Janice Robertson devised a new method based on liposome extrusion and single-molecule fluorescence photobleaching analysis to accurately measure the dimer association free energy of a ClC-type chloride ion/hydrogen ion antiporter.  (Janice started this work when she was a postdoc in Chris Miller’s lab at Brandeis and later completed the project in her own  lab at the University of Iowa.)  The study reveals that ClC-ec1 “is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.”

The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers
Rahul Chadda, Venkatramanan Krishnamani, Kacey Mersch, Jason Wong, Marley Brimberry, Ankita Chadda, Ludmila Kolmakova-Partensky, Larry J Friedman, Jeff Gelles, and Janice L Robertson
eLife (2016) 5:e17438

Timothy Harden, Ph.D.

Congratulations! to Tim Harden, who successfully defended his Ph.D. dissertation in Physics with an additional specialization in Quantitative Biology.  Tim was jointly advised by Jeff Gelles and Jane Kondev.  He is now a Postdoctoral Fellow in Angela DePace’s lab at Harvard Medical School.

Jeff, Tim, Jane

L-to-R: Jeff, Tim, Jane

“Single molecule analysis reveals reversible and irreversible steps during spliceosome activation”

“The spliceosome is a complex molecular machine, composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins, that excises introns from precursor messenger RNAs (pre-mRNAs).  After assembly, the spliceosome is activated for catalysis by rearrangement of subunits to form an active site.” This study used multi-wavelength single-molecule fluorescence (“CoSMoS”) techniques to elucidate the mechanism of budding yeast spliceosome activation.  Activation turns out to be unexpectedly dynamic and variable: some spliceosomes  take multiple attempts to activate and the pathway contains both reversible and irreversible steps.  Strikingly, ATP powers both steps that drive the process forward toward splicing and well as reverse steps that diassemble intermediates to allow subsequent re-attempts at activation. These findings give new insight into how the efficiency and fidelity of pre-mRNA splicing is maintained.Predominant spliceosome activation pathway

The scientific project in this paper was initiated by Aaron Hoskins during his postdoctoral work in Melissa Moore’s and Jeff Gelles’ labs, but it was brought to fruition by Aaron and Margaret Rodgers working in Aaron’s lab at Univ. Wisconsin, Madison.

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
Aaron A. Hoskins Margaret L. Rodgers , Larry J. Friedman , Jeff Gelles , Melissa J. Moore
eLife (2016) 5:e14166

Radcliffe Fellow

During Jeff’s sabbatical in 2016-17, he will be a Fellow at the Radcliffe Institute for Advanced Study at Harvard University, working on a project to study eukaryotic mRNA synthesis and matuaration mechanisms using single-molecule fluorescence methods.  In addition to this new project he will also be spending time each week at Brandeis working with students and other scientists on the lab’s ongoing projects including those supported by NIH and the Mathers Foundation.  (The lab will be accepting Ph.D. students for rotation projects during 2016-17.)

Grant renewal: Molecular mechanisms coordinating the actin and microtubule cytoskeletons

The National Institute of General Medical Sciences, National Institutes of Health, has awarded a four-year renewal of “Molecular mechanisms coordinating the actin and microtubule cytoskeletons”. This grant funds a joint research project of Jeff Gelles’ and Bruce Goode’s labs at Brandeis.  The project is to determine the molecular processes by which microtubule and actin networks interact to control the architecture and dynamics of eukaryotic cells.  The project has resulted in numerous publications co-authored by members of the two labs, and we are grateful for the opportunity to continue this research.nigms_logo

“Bacterial RNA polymerase can retain σ70 throughout transcription”

“In all kingdoms of life, gene transcription is not carried out by RNA polymerase enzymes alone.” Instead, accessory proteins ride along with RNA polymerase molecules as the latter move along a gene, regulating their biological function and controlling gene expression. However, in no cases is the kinetic mechanism of such elongation regulation quantitatively understood.

Sigma proteins are known to be regulators of bacterial transcription initiation. However, previous work suggested that σ70 is present on some transcription elongation complexes, although the extent to which it is retained from initiation, how long it remains attached, and its consequences for transcription regulation were unclear. In this study, Tim Harden and his collaborators used a novel multi-wavelength single-molecule fluorescence microscopy approach to directly observe and quantitatively characterize the dynamic interactions of the σ70 protein with bacterial RNA polymerase molecules in vitro during active RNA synthesis. Harden is a Brandeis Physics Ph.D. student who is jointly advised by Jeff Gelles and Jane Kondev.  The study demonstrates by direct observation that actively elongating polymerase molecules can retain σ70 from initiation into the elongation phase of transcription; shows that retained σ70 subunits dissociate so slowly that most are still present on the elongation complex at the end of a long gene; and proves that only the subpopulation of elongating polymerases with bound σ70 recognize a class of transcriptional pause sequences which in some contexts play a well-established role in regulating gene expression.hardenfig

More generally, this study provides the first quantitative framework that defines the post-initiation roles of σ70, information that is essential to the understanding of global transcription regulation in bacteria. Furthermore, the work demonstrates a general method for elucidating the dynamic interactions of transcription factors with active elongation complexes; this method has broad application in both prokaryotic and eukaryotic transcription biology.

Bacterial RNA polymerase can retain σ70 throughout transcription
Timothy T. Harden, Christopher D. Wells, Larry J. Friedman, Robert Landick, Ann Hochschild, Jane Kondev, and Jeff Gelles
PNAS (2016) 113:602-607

Resources: Plasmids described in this article are available from Addgene.