In vitro display of DNA elements that modulate the RNA transcription in cis or in trans directions

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.

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Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- and filament-dependent manner

Molecular Biology of the Cell ◽

10.1091/mbc.e19-07-0362 ◽

2019 ◽

Vol 30 (23) ◽

pp. 2913-2928 ◽

Cited By ~ 6

Author(s):

Konstantinos Nakos ◽

Megan R. Radler ◽

Elias T. Spiliotis

Keyword(s):

Membrane Trafficking ◽

Binding Proteins ◽

Live Cells ◽

Dependent Manner ◽

Guanosine Diphosphate ◽

In Vitro Reconstitution ◽

In Trans ◽

In Cis ◽

Precise Role

Septins (SEPTs) are filamentous guanosine-5′-triphosphate (GTP)-binding proteins, which affect microtubule (MT)-dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus-end growth and elongation, while higher and intermediate concentrations inhibit and pause plus-end growth, respectively. We show that SEPT2/6/7 has a modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5′- O-[γ-thio]triphosphate (GTPγS)-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus-end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus-end growth and the tracking of plus end–binding proteins and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment. [Media: see text]

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Cis and trans RET signaling control the survival and central projection growth of rapidly adapting mechanoreceptors

eLife ◽

10.7554/elife.06828 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 16

Author(s):

Michael S Fleming ◽

Anna Vysochan ◽

Sόnia Paixão ◽

Jingwen Niu ◽

Rüdiger Klein ◽

...

Keyword(s):

Cell Survival ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Developmental Process ◽

Central Projection ◽

Drg Neurons ◽

In Trans ◽

Cis And Trans ◽

In Cis

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.

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Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis

Journal of Experimental Medicine ◽

10.1084/jem.20050125 ◽

2005 ◽

Vol 201 (10) ◽

pp. 1627-1635 ◽

Cited By ~ 189

Author(s):

Yok-Ai Que ◽

Jacques-Antoine Haefliger ◽

Lionel Piroth ◽

Patrice François ◽

Eleonora Widmer ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Binding Domain ◽

Disease Evolution ◽

Cell Internalization ◽

Fibrinogen Binding ◽

In Trans ◽

Fibronectin Binding ◽

In Cis

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

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R-Loop FormationIn Transat an AGGAG Repeat

Journal of Nucleic Acids ◽

10.1155/2013/629218 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Kazuya Toriumi ◽

Takuma Tsukahara ◽

Ryo Hanai

Keyword(s):

Chemical Modification ◽

Rna Polymerase ◽

T7 Rna Polymerase ◽

Loop Formation ◽

Ribonuclease T1 ◽

In Trans ◽

Negative Supercoiling ◽

In Cis

Formation of RNA-DNA hybrid, or R-loop, was studiedin vitroby transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formationin ciswas diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loopin transwith DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling.

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A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN

Nucleic Acids Research ◽

10.1093/nar/gkaa1224 ◽

2020 ◽

Author(s):

Shubo Zhao ◽

Anja Kieser ◽

Hao-Yi Li ◽

Hannah K Reinking ◽

Pedro Weickert ◽

...

Keyword(s):

Genome Instability ◽

Negative Control ◽

Premature Aging ◽

Independent Manner ◽

Autocatalytic Cleavage ◽

In Trans ◽

Protein Components ◽

In Cis ◽

Protein Crosslinks

Abstract Repair of covalent DNA–protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.

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Molecular contortionism – on the physical limits of serpin ‘loop-sheet’ polymers

Biological Chemistry ◽

10.1515/bc.2010.085 ◽

2010 ◽

Vol 391 (8) ◽

Cited By ~ 12

Author(s):

James A. Huntington ◽

James C. Whisstock

Keyword(s):

Proper Function ◽

Alternative Mechanism ◽

Polymer Properties ◽

Reactive Centre Loop ◽

Partial Unfolding ◽

In Trans ◽

In Cis ◽

Β Sheet

Abstract Members of the serpin (serine protease inhibitor) superfamily fold into a metastable conformation that is crucial for proper function. As a consequence, serpins are susceptible to mutations that cause misfolding and the intracellular accumulation of pathogenic polymers. The mechanism of serpin polymerisation remains to be resolved, however, over the past two decades the ‘loop-sheet’ hypothesis has gained wide acceptance. In this mechanism the reactive centre loop of one serpin monomer inserts into the β-sheet A of another (in trans), in a manner similar to what is seen for reactive centre loop-cleaved and latent conformations (in cis). The hypothesis has been refined in response to certain experimental data, but it has proved difficult to assess the various propositions without creating molecular models. Here we evaluate the loop-sheet mechanism by creating models of pentamers of the archetypal serpin α1-antitrypsin. We conclude that an inescapable consequence of the loop-sheet mechanism is polymer compaction and rigidity, properties that are inconsistent with the ‘beads-on-a-string’ morphology of polymers obtained from human tissue. The recent crystal structure of a domain-swapped serpin dimer suggests an alternative mechanism that is consistent with known polymer properties, including the requirement of partial unfolding to induce polymer formation in vitro, and polymerisation from a folding intermediate in vivo.

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Septin 2/6/7 complexes tune microtubule plus end growth and EB1 binding in a concentration- and filament-dependent manner

10.1101/638296 ◽

2019 ◽

Author(s):

Konstantinos Nakos ◽

Megan R. Radler ◽

Elias T. Spiliotis

Keyword(s):

Membrane Trafficking ◽

Actin Filaments ◽

Binding Proteins ◽

Live Cells ◽

Dependent Manner ◽

In Vitro Reconstitution ◽

In Trans ◽

In Cis ◽

Precise Role

AbstractSeptins are filamentous GTP-binding proteins, which affect microtubule (MT) dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus end growth and elongation, while higher and intermediate concentrations inhibit and pause plus end growth, respectively. We show that SEPT2/6/7 has a 1.5-fold preference for GTP-over GDP-bound MT lattice, and competes with EB1 for binding to GTPγS-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus end growth and the tracking of plus end-binding proteins, and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment.Highlight Summary for eTOCKnowledge of septin roles in MT dynamics is poor and confounded by knockdown studies. Here, in vitro reconstitution assays show concentration-dependent effects of SEPT2/6/7 on MT plus end growth, pausing and EB1 tracking. We found that SEPT2/6/7 filaments are potent than actin in pausing MT growth and dissociating EB1 from intersecting plus ends.

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The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence

Biochemical Journal ◽

10.1042/bj3190351 ◽

1996 ◽

Vol 319 (2) ◽

pp. 351-359 ◽

Cited By ~ 47

Author(s):

Hans-Dietmar BEER ◽

Gerd WOHLFAHRT ◽

Rolf D. SCHMID ◽

John E. G. McCARTHY

Keyword(s):

Amino Acids ◽

Mutational Analysis ◽

Rhizopus Oryzae ◽

Direct Synthesis ◽

Cysteine Residue ◽

Extracellular Lipase ◽

Catalytic Role ◽

In Trans ◽

In Cis

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position -68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.

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