scholarly journals A microtubule dynamics reconstitutional convention

2016 ◽  
Vol 215 (3) ◽  
pp. 305-307
Author(s):  
Kevin C. Slep
Keyword(s):  
Kinase Activity ◽  
Biological Process ◽  
Microtubule Dynamics ◽  
The Core ◽  
Polo Kinase ◽  
Cell Biol ◽  
In Vitro Reconstitution ◽  
Core Components

In vitro reconstitution is the fundamental test for identification of the core components of a biological process. In this issue, Moriwaki and Goshima (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201604118) reconstitute all phases of microtubule dynamics through the inclusion of five key regulators and demonstrate that Polo kinase activity shifts the system from an interphase mode into an enhanced mitotic mode.

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

scholarly journals A Fully Recombinant System for Activator-dependent Archaeal Transcription

2004 ◽  
Vol 279 (50) ◽  
pp. 51719-51721 ◽  
Author(s):  
Mohamed Ouhammouch ◽  
Finn Werner ◽  
Robert O. J. Weinzierl ◽  
E. Peter Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Regulators ◽  
The Core ◽  
General Transcription Factor ◽  
Core Components ◽  
Archaeal Transcription ◽  
Bacterial Type

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from thein vitroassembly of the 12-subunit RNA polymerase from the hyperthermophileMethanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

In Vitro Reconstitution of the Core and Peripheral Light-Harvesting Complexes ofRhodospirillummolischianumfrom Separately Isolated Components†

Biochemistry ◽  
1998 ◽  
Vol 37 (50) ◽  
pp. 17458-17468 ◽  
Author(s):  
John B. Todd ◽  
Pamela S. Parkes-Loach ◽  
Joseph F. Leykam ◽  
Paul A. Loach
Keyword(s):  
Light Harvesting ◽  
Light Harvesting Complexes ◽  
The Core ◽  
In Vitro Reconstitution

scholarly journals In Vitro Reconstitution and Imaging of Microtubule Dynamics by Fluorescence and Label-free Microscopy

2020 ◽  
Vol 1 (3) ◽  
pp. 100177
Author(s):  
William Graham Hirst ◽  
Christine Kiefer ◽  
Mohammad Kazem Abdosamadi ◽  
Erik Schäffer ◽  
Simone Reber
Keyword(s):  
Microtubule Dynamics ◽  
Label Free ◽  
In Vitro Reconstitution

scholarly journals Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

scholarly journals Autoregulatory control of microtubule binding in the oncogene, doublecortin-like kinase 1

2020 ◽  
Author(s):  
Melissa M. Rogers ◽  
Amrita Ramkumar ◽  
Ashlyn M. Downing ◽  
Hannah Bodin ◽  
Julia Castro ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Binding Activity ◽  
Unique Form ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
In Vitro Reconstitution ◽  
Protein Map

AbstractThe microtubule-associated protein (MAP), doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for the development of kinase inhibitors. However, the physiological roles of its kinase activity and how DCLK1 kinase activity is regulated remain elusive. Here we employ in vitro reconstitution with purified proteins to analyze the role of DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a single residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation largely within the doublecortin 2 (DC2) domain, which dramatically reduces the microtubule affinity of DCLK1. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule’s ability to associate with microtubules. Overall, our results suggest a mechanism by which DCLK1 modulates its own kinase activity to tune its microtubule binding affinity, providing molecular insights into a unique form of autoregulatory control over microtubule binding activity within the broader family of MAPs. These results provide useful molecular insights for future therapeutic efforts related to DCLK1’s role in cancer development and progression.

scholarly journals Structural mimicry confers robustness in the cyanobacterial circadian clock

2020 ◽  
Author(s):  
Joel Heisler ◽  
Jeffrey A. Swan ◽  
Joseph G. Palacios ◽  
Cigdem Sancar ◽  
Dustin C. Ernst ◽  
...  
Keyword(s):  
Gene Expression ◽  
Circadian Rhythms ◽  
Histidine Kinase ◽  
Kinase Activity ◽  
Protein Complexes ◽  
Competitive Interactions ◽  
The Core ◽  
Oscillator Function ◽  
Clock Protein

AbstractThe histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC. Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness.One Sentence SummarySasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

scholarly journals Aptamers and Possible Effects on Neurodegeneration

2020 ◽  
Author(s):  
Fatma Söylemez ◽  
Çağatay Han Türkseven
Keyword(s):  
Thermal Stability ◽  
Medical Equipment ◽  
Low Cost ◽  
Tissue Samples ◽  
New Class ◽  
High Affinity ◽  
The Core ◽  
Core Components ◽  
Near Future

Aptamers are a new class of recognizing agents which are defined as short biomolecules like oligonucleotides and peptides that are used in diagnostics and therapeutics. They can bind to specific targets with extremely high affinity based on their structural conformations. It is believed that in the near future, aptamers could replace monoclonal antibody. The biggest advantage of using aptamers is that the process is in vitro in nature and does not require the use of animals and they also have unique properties, such as thermal stability, low cost, and unlimited applications. Aptamers have been studied as a biomaterial in numerous investigations concerning their use as a diagnostic and therapeutic tool and biosensing probe. DNA aptamers were also used for the diagnosis and treatment of neurodegeneration and neurodegenerative diseases. For example, functional nucleic acid aptamers have been developed to detect Aβ fragments in Alzheimer’s brain hippocampus tissue samples. Aptamers are promising materials for diverse areas, not just as alternatives to antibodies but as the core components of medical equipment. Although they are in the preliminary stages of development, results are quite encouraging, and it seems that aptamer research has a very bright future in neuroscience.

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