Grp94 is a molecular chaperone that helps to fold and maintain the folded state of “client” proteins in the endoplasmic reticulum. Acceleration of client folding is driven by conformational changes in Grp94. However, the sequence of conformational changes and how these changes are coupled to the cycle of ATP hydrolysis is not well understood. Prof. Timothy Street and his lab members Bin Huang and Ming Sun, in collaboration with Larry Friedman, did single-molecule fluorescence resonance energy transfer (FRET) experiments to directly observe conformational cycling in individual Grp94 molecules. Their studies show that ATP hydrolysis can drive repeated cycling between alternative “closed” states of Grp94, suggesting a way that enzyme might propagate structural changes to client molecules.
Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone
Huang, B., Friedman, L.J., Gelles, J., Sun, M., and Street, T.O.
eLife (2019) 8:e40576
RNA polymerases contain a conserved “secondary channel” in which proteins that regulate transcription can bind. In Escherichia coli bacteria, the secondary channel factors (SCFs) GreB and DksA both repress initiation of ribosomal RNA synthesis, but SCF loading and repression mechanisms are unclear. Sarah Stumper and her collaborators used fluorescence correlation spectroscopy and multi-wavelength single-molecule fluorescence colocalization microscopy to show that the SCFs likely repress transcription through an interesting “delayed inhibition” mechanism in which the proteins arrive at DNA already complexed to RNA polymerase and block at a later stage of transcription initiation. The work explains factors that control the relative contributions of the two proteins to regulation and suggests a mechanism by which repression is restricted to ribosomal RNA and other promoters that form short-duration complexes with RNA polymerase.
Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription.
Stumper, S.K., Ravi, H., Friedman, L.J., Mooney, R.A., Corrêa, I.R., Gershenson, A., Landick, R., and Gelles, J.
eLife (2019) 8:e40576
Branched actin filament networks formed by the Arp2/3 complex play an essential role in force production in eukaryotic cells. Branched networks are not static components of the cytoskeleton. Instead the times and locations of network assembly and disassembly are tightly controlled by regulatory proteins. Ph.D. student Siyang Guo used single-molecule fluorescence methods to show how the Abp1 protein positively regulates branched actin networks. Remarkably, Apb1 functions by two distinct mechanisms. The protein stimulates the formation of networks by stabilizing the binding of Arp2/3 complex to the sides of actin filaments, a precursor to branch formation. However after branches form bound Abp1 works differently: it protects the network from GMF, the “pruning shears” protein that chops off branches during network disassembly. Taken as a whole, the study gives deeper insight into the multiple layers of regulation that control cytoskeleton pattern formation and dynamics. This project is part of a long-term collaboration on cytoskeletal regulation with Bruce Goode’s lab.
Abp1 promotes Arp2/3 complex-dependent actin nucleation and stabilizes branch junctions by antagonizing GMF
Siyang Guo, Olga S. Sokolova, Johnson Chung, Shae Padrick, Jeff Gelles, Bruce L. Goode
Nature Communications (2018) 9:2895.
In human genes, protein-coding regions alternate with non-coding “introns” that must be snipped out of the RNA transcript before it is used to produce a protein. The snipping is done by the spliceosome, a complex molecular machine that must assemble anew on each intron it removes. The spliceosome must cut out exactly the segments of the messenger molecule that require removal, no more and no less, since inaccurate intron removal can produce a messenger molecule that is non-functional or that causes disease.
To help understand how multiple introns are quickly and accurately removed, postdoctoral fellow Joerg Braun developed a light microscopy method by which for the first time we can observe the coordinated processes by which human spliceosomes recognize and assemble around the segments of single messenger RNA molecules. As the eLife digest puts it: “The experiments show that spliceosomes working on different introns in the same pre-mRNA actually help each other out. As one assembles, this helps the spliceosome that processes the neighboring intron to get built. In particular, the U1snRNPs [a spliceosome sub-assembly] processing nearby introns collaborate to promote the assembly and activity of the spliceosomes. This teamwork is likely important to guarantee that multiple introns are cut out quickly and accurately.”
Synergistic assembly of human pre-spliceosomes across introns and exons.
Joerg E. Braun, Larry J. Friedman, Jeff Gelles, and Melissa J. Moore.
eLife (2018) 7:e37751.
New plasmids reported in this article can be obtained from Addgene.
Computer software used in this research can be obtained from GitHub.
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.
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.
New plasmids reported in this article can be obtained from Addgene
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
“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.
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
“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.
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.
Regulation of actin filament length is a central process by which eukaryotic cells control the shape, architecture, and dynamics of their actin networks. This regulation plays a fundamental role in cell motility, morphogenesis, and a host of processes specific to particular cell types. This paper by recently graduated Ph.D. student Jeffrey Bombardier and collaborators resolves the long-standing mystery of how formins and capping protein work in concert and antagonistically to control actin filament length. Bombardier used the CoSMoS multi-wavelength single-molecule fluorescence microscopy technique to to discover and characterize a novel tripartite complex formed by a formin, capping protein, and the actin filament barbed end. Quantitative analysis of the kinetic mechanism showed that this complex is the essential intermediate and decision point in converting a growing formin-bound filament into a static capping protein-bound filament, and the reverse. Interestingly, the authors show that “mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex.” The results define the essential features of the molecular mechanism of filament length regulation by formin and capping protein; this mechanism predicts several new ways by which cells are likely to couple upstream regulatory inputs to filament length control.
Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end
Jeffrey P. Bombardier, Julian A. Eskin, Richa Jaiswal, Ivan R. Corrêa, Jr., Ming-Qun Xu, Bruce L. Goode, and Jeff Gelles
Nature Communications 6:8707 (2015)
Resources: The capping protein expression plasmid described in this article is available from Addgene.
Readers interested in this subject should also see a related article by Shekhar et al published simultaneously in the same journal. We are grateful to the authors of that article for coordinating submission so that the two articles were published together.