Journal ArticleDOI
Kinetic mechanism of nucleotide binding toEscherichia coli transcription termination factor Rho: Stopped-flow kinetic studies using ATP and fluorescent ATP analogues
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TLDR
Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and Dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.Abstract:
Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2’(3’)-O-(N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6×106 M−1sec−1, whereas the second nucleotide binds at a slower rate of 4.7×105 M−1 sec−1 at 18°C. RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.read more
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Journal ArticleDOI
New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.
TL;DR: The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.
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Transcription termination factor rho is an RNA-DNA helicase
TL;DR: The results suggest that NTP hydrolysis is utilized to help unwind the RNA-DNA duplex at the 3' end of a nascent transcript, facilitating RNA release from the DNA template.
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A structural model for the Escherichia coli DnaB helicase based on electron microscopy data.
TL;DR: Electron microscopic images of negatively stained DnaB protein have been studied and processed to produce a three-dimensional reconstruction of the protein oligomer and a structural model for the oligomer is presented, and functional implications are considered.
Journal ArticleDOI
The hexameric E. coli DnaB helicase can exist in different Quaternary states.
TL;DR: The DnaB protein is the primary replicative helicase in Escherichia coli, and the active form of the protein is a hexamer, and it is found that both forms are hexamers, excluding the possibility that the 3-fold form is in fact a trimer of the 52 kDa monomer.
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The structural basis for terminator recognition by the Rho transcription termination factor.
TL;DR: Dimmerization of symmetric subunits on an asymmetric ligand is developed as a model for allosteric control in the action of the intact Rho hexamer, suggesting that the strongRNA-binding sites of Rho may arise from pairing of RNA-binding modules.