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6. Microtubule-based active fluids with improved lifetime, temporal stability and miscibility with passive soft materials

Author

Chandrakar, Pooja, Berezney, John, Lemma, Bezia, Hishamunda, Bernard, Berry, Angela, Wu, Kun-Ta, Subramanian, Radhika, Chung, Johnson, Needleman, Daniel, Gelles, Jeff, and Dogic, Zvonimir

Subjects
Condensed Matter - Soft Condensed Matter, Physics - Biological Physics
Abstract

We have developed several distinct model systems of microtubule-based 3D active isotropic fluids and have compared their dynamical and structural properties. The non-equilibrium dynamics of these fluids is powered by three different types of kinesin motors: a processive motor, a non-processive motor, and a motor which is permanently linked to a microtubule backbone. The fourth modification uses specific microtubule crosslinkers to induce bundle formation instead of a non-specific depletant. In comparison to the formerly developed systems, each new active fluid formulation has certain enhanced properties. Firstly, linking motors to the microtubule backbone enhances the fluid lifetime from hours to several days. Furthermore, switching to the non-processive motors significantly increases the temporal stability of the active dynamics, while using specific cross-linkers yields systems that can incorporate other passive soft materials, such as, polymer gels or liquid crystals. These novel developed model systems will significantly aid and improve our ability to quantify diverse phenomena observed in microtubule based active matter., Comment: 14 pages, 11 figures

Published
2018

17. Source data for 'Recycling of Bacterial RNA Polymerase by the Swi2/Snf2 ATPase RapA'

Author

Inlow, Koe, Tenenbaum, Debora, Friedman, Larry J., Kondev, Jane, and Gelles, Jeff

Abstract

Source data for: Recycling of Bacterial RNA Polymerase by the Swi2/Snf2 ATPase RapA In press, PNAS 2023 Koe Inlowa, Debora Tenenbaumb, Larry J. Friedmana, Jane Kondevb,*, and Jeff Gellesa,* aDepartment of Biochemistry and bDepartment of Physics, Brandeis University, Waltham, MA 02453, USA. *To whom correspondence may be addressed. Email: gelles@brandeis.edu (J.G.) or kondev@brandeis.edu (J.K.). Source data for the single-molecule experiments is provided as “intervals” files that can be read and manipulated by the Matlab program “Imscroll”, which is publicly available at: https://github.com/gelles-brandeis/CoSMoS_Analysis The files are identified in Source data index.docx, the contents of which are also reproduced below. --------------------------------------------------------------------- Fig 1B, 1E (top): ki00447_core_intervals.dat; ki00446_oligo_intervals.dat ki00174_core_intervals.dat; ki00175_oligo_intervals.dat ki00586_core_intervals.dat; ki00587_oligo_intervals.dat Fig 1C, 1D, 1E (bottom): ki00411_core_intervals.dat; ki00411_RapA_intervals.dat; ki00412_oligo_intervals.dat ki00416_core_intervals.dat; ki00416_RapA_intervals.dat; ki00417_oligo_intervals.dat ki00590_core_intervals.dat; ki00592_RapA_intervals.dat; ki00591_oligo_intervals.dat Fig 2B: Fig 2C, premix (orange data): 0 nM RapA, no ATP: ki00038_intervals.dat 5 nM RapA, no ATP: ki00022_intervals.dat; ki00007_intervals.dat 10 nM RapA, no ATP: ki00083_intervals.dat; ki00031_intervals.dat 0 nM RapA + 1 mM ATP: ki00026_intervals.dat; ki00117_intervals.dat 1 nM RapA + 1 mM ATP: ki00140_core_intervals.dat; ki00010_intervals.dat 5 nM RapA + 1 mM ATP: ki00136_intervals.dat; ki00164_core_intervals.dat; ki00044_intervals.dat 10 nM RapA + 1 mM ATP: ki00016_intervals.dat; ki00034_intervals.dat; ki00123_intervals.dat; ki000444_intervals.dat 25 nM RapA + 1 mM ATP: ki00218_core_intervals.dat 50 nM RapA + 1 mM ATP: ki00225_core_intervals.dat 100 nM RapA + 1 mM ATP: ki00047_intervals.dat 500 nM RapA + 1 mM ATP: ki000381_intervals.dat; ki00042_intervals.dat; ki00050_intervals.dat Fig 2C, washout (green data): 0 nM RapA, no ATP: ki00092_intervals.dat; ki00254_intervals.dat; ki00075_intervals.dat 2 nM RapA, no ATP: ki00058_intervals.dat 4 nM RapA, no ATP: ki00355_core_intervals.dat 5 nM RapA, no ATP: ki00287_intervals.dat; ki00294_intervals.dat; ki00313_intervals.dat; ki00327_core_intervals.dat; ki00381_core_intervals.dat 50 nM RapA, no ATP: ki00474_core_intervals.dat 75 nM RapA, no ATP: ki00246_intervals.dat; ki00304_intervals.dat; ki00081_intervals.dat 100 nM RapA, no ATP: ki00290_intervals.dat; ki00065_intervals.dat 0 nM RapA + 1 mM ATP: ki00344_core_intervals.dat; ki00236_intervals.dat 1 nM RapA + 1 mM ATP: ki00015_intervals.dat; ki00263_intervals.dat; ki00279_intervals.dat; ki00331_core_intervals.dat; ki00378_core_intervals.dat; ki00429_core_intervals.dat 2 nM RapA + 1 mM ATP: ki00478_core_intervals; ki00267_intervals.dat 2.5 nM RapA + 1 mM ATP: ki00335_core_intervals.dat; ki00482_core_intervals.dat; ki00318_intervals.dat 3 nM RapA + 1 mM ATP: ki00283_core_intervals.dat; ki00341_core_intervals.dat; ki00465_core_intervals.dat 5 nM RapA + 1 mM ATP: ki00017_intervals.dat; ki00298_core_intervals.dat; ki00301_core_intervals.dat; ki00271_intervals.dat; ki00288_core_intervals.dat; ki00323_core_intervals.dat 10 nM RapA + 1 mM ATP: ki00084_intervals.dat; ki00240_core_intervals.dat Fig 3A: ki00491_RapA_intervals.dat (ki00491_RapA_intervals_new_all_spots_high_frmavg2_frames1to3005_newmap_rad2point4_up2.dat) ki00491_core_intervals.dat (ki00491_core_intervals_frmavg2_frames1_to_3005_newmap_rad2_up2point1.dat) ki00507_RapA_intervals.dat (ki00507_RapA_intervals_bg_corrected_frmavg2_newmap_rad2point5_up1point5.dat) ki00507_core_intervals.dat (ki00507_core_intervals_newmapfile_rad2_up1point5.dat) Fig 3B: RapA binding npDNA (orange): ki00573_RapA_intervals.dat RapA binding off-target npDNA (grey): ki00573_RapA_off_target_intervals.dat RapA binding rPTCs (blue): ki00491_RapA_intervals.dat RapA binding off-target rPTCs (black): ki00491_RapA_off_target_intervals.dat (ki00491_RapA_off_target_intervals_up1point2_rad1.dat) Fig 3C: ki00513_core_intervals.dat (ki00513_core_intervals_bg_corrected_uprad2.dat) ki00513_RapA_intervals.dat (ki00513_RapA_intervals_bg_corrected_uprad2.dat) ki00510_core_intervals.dat (ki00510_core_intervals_bg_corrected_rad2point5_up2point1.dat) ki00510_RapA_intervals.dat (ki00510_RapA_intervals_bg_corrected_rad3_up1point5.dat) Fig 3D: ki00510_core_intervals.dat (ki00510_core_intervals_bg_corrected_rad2point5_up2point1.dat) ki00510_RapA_intervals.dat (ki00510_RapA_intervals_bg_corrected_rad3_up1point5.dat) Fig 4A, S6: No nucleotide, green: ki00551_core_intervals.dat (ki00551_core_intervals_uprad2.dat) No nucleotide, red: ki00551_RapA_intervals.dat (ki00551_RapA_intervals_rad2_up1point5.dat) No nucleotide, blue: ki00552_RapA_intervals.dat (ki00552_RapA_intervals_rad2_up1point5.dat) + 1 mM AMPPNP, green: ki00555_core_intervals.dat (ki00555_core_intervals_uprad2_frmavg2.dat) + 1 mM AMPPNP, red: ki00555_RapA_intervals.dat (ki00555_RapA_intervals_uprad2_frmavg2.dat) + 1 mM AMPPNP, blue: ki00556_RapA_intervals.dat (ki00556_RapA_intervals_rad2_up2point5_frmavg2.dat) + 1 mM ATP, green: ki00559_core_intervals.dat (ki00559_core_intervals_uprad2.dat) + 1 mM ATP, red: ki00559_RapA_intervals.dat (ki00559_RapA_intervals_up1point5_rad2point5.dat) + 1 mM ATP, blue: ki00560_RapA_intervals.dat (ki00560_RapA_intervals_uprad2.dat) Fig 4B-C, 5A: + 1 mM AMPPNP (purple): ki00555_RapA_intervals.dat; ki00555_RapA_off_target_intervals.dat (ki00555_RapA_off_target_intervals_uprad2.dat) + 1 mM ATP (blue): ki00559_RapA_intervals.dat; ki00559_RapA_off_target_intervals.dat ki00559_RapA_off_target_intervals_up1point5_rad2point5.dat No nucleotide (grey): ki00551_RapA_intervals.dat; ki00551_RapA_off_target_intervals.dat (ki00551_RapA_off_target_intervals_rad2_up1point5.dat) Fig S1: Black: ki00295_intervals.dat (ki00236_intervals_rad2point5_thres6.dat) Blue: ki00298_intervals.dat Purple: ki00301_intervals.dat Fig S3: 250 uW: ki00236_intervals.dat 350 uW: ki00254_intervals.dat 500 uW: ki00344_core_intervals.dat 1500 uW: ki00597_intervals.dat Fig S4: 1080 uW: ki00516_RapA_intervals.dat (ki00516_RapA_intervals_frmavg2_bg_corrected_uprad2point5.dat) 5250 uW: ki00494_RapA_intervals.dat (ki00494_RapA_intervals_rad2point5_up1point5.dat) 321 uW: ki00556_RapA_intervals.dat &nbsp

Published
2023
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29. RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons.

Author

Tenenbaum, Debora, Inlow, Koe, Friedman, Larry J., Cai, Anthony, Gelles, Jeff, and Kondev, Jane

Subjects
BACTERIAL operons, RNA polymerases, DNA polymerases, BACTERIAL genomes, MESSENGER RNA
Abstract

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ70; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator–promoter distances up to ∼1,000 bp, which includes the majority of terminator–promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA. [ABSTRACT FROM AUTHOR]

Published
2023
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30. Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.

Author

Amasino, Audra L., Gupta, Shalini, Friedman, Larry J., Gelles, Jeff, and Bell, Stephen P.

Subjects
PHOSPHORYLATION, DNA replication, CELL cycle proteins, CELL cycle, PLOIDY
Abstract

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation. [ABSTRACT FROM AUTHOR]

Published
2023
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31. Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA.

Author

Inlow, Koe, Tenenbaum, Debora, Friedman, Larry J., Kondev, Jane, and Gelles, Jeff

Subjects
BACTERIAL RNA, RNA polymerases, ADENOSINE triphosphatase, SINGLE molecules, FLUORESCENCE microscopy
Abstract

Free- living bacteria have regulatory systems that can quickly reprogram gene transcrip- tion in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)—consisting of core RNA polymerase (RNAP)- bound sequence nonspecifically to double-stranded DNA—and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis- dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes. [ABSTRACT FROM AUTHOR]

Published
2023
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37. A helicase-tethered ORC flip enables bidirectional helicase loading

Author

Massachusetts Institute of Technology. Department of Biology, Gupta, Shalini, Friedman, Larry J, Gelles, Jeff, Bell, Stephen P, Massachusetts Institute of Technology. Department of Biology, Gupta, Shalini, Friedman, Larry J, Gelles, Jeff, and Bell, Stephen P

Abstract

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin–recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7–ORC–DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.

Published
2022

49. Alternative transcription cycle for bacterial RNA polymerase

Author

Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Physics, Harden, Timothy T, Herlambang, Karina S, Chamberlain, Mathew, Lalanne, Jean-Benoît, Wells, Christopher D, Li, Gene-Wei, Landick, Robert, Hochschild, Ann, Kondev, Jane, Gelles, Jeff, Massachusetts Institute of Technology. Department of Biology, Massachusetts Institute of Technology. Department of Physics, Harden, Timothy T, Herlambang, Karina S, Chamberlain, Mathew, Lalanne, Jean-Benoît, Wells, Christopher D, Li, Gene-Wei, Landick, Robert, Hochschild, Ann, Kondev, Jane, and Gelles, Jeff

Abstract

© 2020, The Author(s). RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

Published
2021

50. DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

Author

Massachusetts Institute of Technology. Department of Biology, Howard Hughes Medical Institute, De Jesús-Kim, Lorraine, Friedman, Larry J, Lõoke, Marko, Ramsoomair, Christian K, Gelles, Jeff, Bell, Stephen P, Massachusetts Institute of Technology. Department of Biology, Howard Hughes Medical Institute, De Jesús-Kim, Lorraine, Friedman, Larry J, Lõoke, Marko, Ramsoomair, Christian K, Gelles, Jeff, and Bell, Stephen P

Abstract

© 2021, eLife Sciences Publications Ltd. All rights reserved. The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

Published
2021

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