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
Jeff Gelles’ and Douglas Theobald’s laboratories were just awarded a grant from NIGMS to develop new statistics-based methods for deducing the mechanisms of biochemical processes from single-molecule fluorescence data.
We have 1-2 postdoctoral positions on this project available for Ph.D. scientists with a strong computation background; for more information see the job announcement.
Jeff will receive the 2019 Kazuhiko Kinosita Award in Single-Molecule Biophysics from the Biophysical Society. Prof. Kinosita was a much-admired pioneer of single-molecule biophysics, famous for his creative and intellectually rigorous approach to science. His research revealed key features of how molecular motors operate and how cells make ATP. Students will enjoy this public lecture from the January 2015 Single Molecule Biophysics conference in which Prof. Kinosita talks about his work:
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 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.
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