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

1996 ◽  
Vol 319 (2) ◽  
pp. 351-359 ◽  
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.

Ty1 in vitro integration: effects of mutations in cis and in trans

1994 ◽  
Vol 14 (9) ◽  
pp. 5731-5740
Author(s):  
L T Braiterman ◽  
J D Boeke
Keyword(s):  
Missense Mutation ◽  
Frameshift Mutations ◽  
Amino Terminal ◽  
Dna Molecule ◽  
Conserved Domain ◽  
In Trans ◽  
In Cis ◽  
Obvious Similarity ◽  
Insertion Mutations

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.

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

Gene ◽  
1996 ◽  
Vol 181 (1-2) ◽  
pp. 135-137
Author(s):  
Barbara Reifenrath-Biesel ◽  
Hinrich Abken
Keyword(s):  
Rna Transcription ◽  
Dna Elements ◽  
In Trans ◽  
In Cis

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

2019 ◽  
Vol 30 (23) ◽  
pp. 2913-2928 ◽  
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]

scholarly journals Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

2004 ◽  
Vol 24 (17) ◽  
pp. 7392-7401 ◽  
Author(s):  
Jan Medenbach ◽  
Silke Schreiner ◽  
Sunbin Liu ◽  
Reinhard Lührmann ◽  
Albrecht Bindereif
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Protein A ◽  
Tetratricopeptide Repeat ◽  
Terminal Sequence ◽  
Recognition Motifs ◽  
Tpr Domain

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

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

eLife ◽  
2015 ◽  
Vol 4 ◽  
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.

Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro

1993 ◽  
Vol 13 (8) ◽  
pp. 4609-4617
Author(s):  
C K Galang ◽  
C A Hauser
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Mutational Analysis ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

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

2005 ◽  
Vol 201 (10) ◽  
pp. 1627-1635 ◽  
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.

scholarly journals Recognition of the Core RNA Promoter for Minus-Strand RNA Synthesis by the Replicases of Brome Mosaic Virus andCucumber Mosaic Virus

2000 ◽  
Vol 74 (22) ◽  
pp. 10323-10331 ◽  
Author(s):  
K. Sivakumaran ◽  
Y. Bao ◽  
M. J. Roossinck ◽  
C. C. Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Direct Synthesis ◽  
Specific Interaction ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Genomic Rnas ◽  
Promoter Recognition

ABSTRACT Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of theBromovirus and Cucumovirus genera have a tRNA-like structure at the 3′ end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. InBrome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5′CA3′ dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

scholarly journals R-Loop FormationIn Transat an AGGAG Repeat

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
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.

scholarly journals Mutational analysis of yeast profilin.

1993 ◽  
Vol 13 (12) ◽  
pp. 7864-7873 ◽  
Author(s):  
B K Haarer ◽  
A S Petzold ◽  
S S Brown
Keyword(s):  
Amino Acids ◽  
Adenylate Cyclase ◽  
Mutational Analysis ◽  
Actin Binding ◽  
In Vitro Assays ◽  
C Terminus ◽  
Physiological Relevance ◽  
In Vivo Effects

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

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