Tag Archives: CoSMoS

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

Schematic representation of DNA replication initiation Replication of chromosomal DNA in eukaryotes has two major stages.  Starting in the G1 phase of the cell cycle, double hexamers consisting of two copies of the Mcm2-7 replicative helicase are assembled at replication origins.   Later, in S phase, the two helicases are incorporated into two oppositely oriented CMG (Cdc45-Mcm2-7-GINS) complexes that each then form the core of a replisome.  Control of this “activation” step, which is triggered by the protein kinases DDK and S-CDK, is essential to ensure that each part of the genome is replicated once and only once in each cell cycle.

In this paper, Lorraine De Jesús-Kim together with collaborators from Steve Bell’s and Jeff Gelles’ labs used multi-wavelength single-molecule fluorescence colocation  (“CoSMoS”) methods to study in vitro the molecular mechanism of the activation process.  The journal’s acceptance summary notes that “The manuscript provides new and convincing evidence that a heretofore unknown intermediate state [called “CtG”] for replication start contains multiple copies of the GINS and Cdc45 proteins prior to initiation at each origin with one double hexamer of the MCM2-7 complex. The number of GINS and Cdc45 is determined by DDK phosphorylation of the MCM’s and the probability to create an active helicase (CMG) is increased with multiple numbers of the bound ancillary factors…. The single molecule studies and biochemistry are beautifully executed providing the evidence for such intermediates…. The addition of in vivo studies demonstrates that modulating the multiplicity of DDK phosphorylation (and proposed, CtG formation) has an impact on origin usage in cells.”Proposed model for Cdc45-Mcm2-7-GINS (CMG) formation

 

10.7554/eLife.65471
Kim L.D.J., et al., DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.
eLife 10, e65471 (2021)

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

DNA transcription by RNA polymerase II (RNApII) is arguably the process most central to regulation of gene expression in eukaryotic organisms.  Regulated transcription requires the formation on DNA of molecular assemblies containing not only RNApII but also dozens of accessory proteins that play pivotal roles in the process.  While we know about the structures of some of these assemblies in atomic detail, quantitative understanding of the dynamics and pathways by which the assemblies interconvert and progress through this fundamental gene expression pathway is largely lacking.

In this study we report single-molecule fluorescence microscopy studies of transcription in yeast nuclear extract, for the first time visualizing and measuring the dynamics of activator-dependent recruitment of RNApII and the central elongation factor Spt4/5 to transcription complexes.  Grace Rosen (Jeff Gelles’ labortatory, Brandeis) , Inwha Baek (Steve, Buratowski’s lab, Harvard Medical School), and collaborators elucidated the kinetically significant steps in activated RNApII transcription initiation and show for the first time that Spt4/5 dynamics are tuned to the typical lifetimes of transcription elongation complexes.  In addition to these substantive results, our work represents an important methodological advance.  As the first application of the CoSMoS (co-localization single-molecule spectroscopy) technique to activated eukaryotic transcription, it demonstrates a general method for elucidating the correlated dynamic interactions of different components of the machinery with initiation and elongation transcription complexes.  The approach is likely to find further use in studies of the mechanistic features of RNApII transcription.

https://doi.org/10.1073/pnas.2011224117
Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
PNAS 

Alternative transcription cycle for bacterial RNA polymerase

DNA transcription is the most important nexus of gene regulation in all living organisms.  In recent years, single-molecule experiments have given us a new window into the mechanisms of transcription and have revealed novel, previously unsuspected molecular behaviors and mechanisms.  Ph.D. student Tim Harden used multi-color single-molecule fluorescence imaging to reveal a completely new transcription cycle for bacterial RNA polymerase.  Harden and co-authors showed that an RNA polymerase molecule in vitro frequently (in >90% of transcription events) remains bound to DNA and may again initiate transcription after it has terminated the first round of transcription.  Even more unexpectedly, this “secondary initiation” is not restricted to the same RNA.  After the first round, the polymerase can scan thousands of basepairs along the DNA and can initiate at a different start site, frequently one that is oriented in the opposite direction and produces an antisense transcript.

cannonical and alternative bacterial transcription cycles

To complement the single-molecule studies in vitro, the manuscript reports new analyses of whole-transcriptome cellular RNAs revealed by the Rend-seq method that measures transcript initiation and termination frequencies across the genome with single basepair resolution.  These provide evidence that the new transcription cycle may be responsible for initiating antisense transcription at hundreds of genomic locations in the two widely divergent bacterial species examined.  The work defines a new mechanism for the regulated production of antisense RNAs, many of which are now recognized as important agents of gene-specific regulation through control of transcription, mRNA decay, and translation.  In addition, the new transcription cycle provides a mechanism through which transcription initiation can be controlled not just through feedback networks involving multiple genes, but also through production of multiple different primary transcripts consequent to a single RNA polymerase-to-DNA recruitment event.

10.1038/s41467-019-14208-9
Harden, T.T., et al. Alternative transcription cycle for bacterial RNA polymerase.
Nature Communications 11, 450 (2020).

Synergy between cyclase-associated protein and cofilin accelerates actin filament depolymerization by two orders of magnitude

From Science at Brandeis:

“All animal and plant cells contain a highly elaborate system of filamentous protein polymers called the actin cytoskeleton, a scaffold that can be rapidly transformed to alter a cell’s shape and function. A critical step in reconfiguring this scaffold is the rapid disassembly (or turnover) of the actin filaments. But how is this achieved? It has long been known that the protein Cofilin plays a central role in this process, but it has been unclear how Cofilin achieves this feat. Cofilin can sever actin filaments into smaller fragments to promote their disassembly, but whether it also catalyzes subunit dissociation from filament ends has remained uncertain and controversial. Until now, this problem has been difficult to address because of limitations in directly observing Cofilin’s biochemical effects at filament ends….”  Dr. Shashank Shekhar, working together with Dr. Johnson Chung and “jointly mentored by Bruce Goode, Jeff Gelles and Jane Kondev, use[d] microfluidics-assisted single molecule TIRF imaging to tackle the problem.

The new study shows that Cofilin and one other protein (Srv2/CAP) intimately collaborate at one end of the actin filament to accelerate subunit dissociation by over 300-fold! These are the fastest rates of actin depolymerization ever observed. Further, these results establish a new paradigm in which a protein that decorates filament sides (Cofilin) works in concert with a protein that binds to filament ends (Srv2/CAP) to produce an activity that is orders of magnitude stronger than the that of either protein alone.”

10.1038/s41467-019-13268-1
Shekhar S. et al. Synergy between cyclase-associated protein and cofilin accelerates actin filament depolymerization by two orders of magnitude.
Nature Communications
10, 5319 (2019).

A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

In this project, Kankowan Champasa from Stephen Bell’s lab at MIT collaborated with other researchers from the Bell and Gelles labs to study a key process that sets the stage for replication of chromosomal DNA. They explain “licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally.”  The researchers identified a conserved motif in the Mcm4 helicase subunit essential for formation of productive replication complexes.  Single-molecule fluorescence energy transfer experiments show that mutations in the motif still allow the two hexamers to come into contact, but they prevent the formation of the stable double-hexamers that perform the extensive DNA unwinding needed for replication.

10.7554/eLife.45538
A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding.
Champasa, K., Blank, C., Friedman, L.J., Gelles, J., and Bell, S.P.
eLife (2019) 8:e40576

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

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.

10.7554/eLife.40576
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

“Synergistic assembly of human pre-spliceosomes across introns and exons”

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.

Implications of synergistic U2 recruitment for the mechanism of exon and intron recognition.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.”

10.7554/eLife.37751
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.
Resources:
New plasmids reported in this article can be obtained from Addgene.
Computer software used in this research can be obtained from GitHub.

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.”

10.1073/pnas.1616525114
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.

Resources:
New plasmids reported in this article can be obtained from Addgene

“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.

10.7554/eLife.14166
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