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Vesicular stomatitis virus polymerase's strong affinity to its template suggests exotic transcription models.

Tang X, Bendjennat M, Saffarian S - PLoS Comput. Biol. (2014)

Bottom Line: Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial.We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies.In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of Utah, Salt Lake City, Utah, United States of America.

ABSTRACT
Vesicular stomatitis virus (VSV) is the prototype for negative sense non segmented (NNS) RNA viruses which include potent human and animal pathogens such as Rabies, Ebola and measles. The polymerases of NNS RNA viruses only initiate transcription at or near the 3' end of their genome template. We measured the dissociation constant of VSV polymerases from their whole genome template to be 20 pM. Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial. To explore possible mechanisms, we simulated the first hour of transcription using Monte Carlo methods and show that a one-time initial dissociation of all polymerases during entry is not sufficient to sustain transcription. We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies. In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.

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Transcription of circular genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a circular genome template, (A) shows a representation of this model with collision rules resulting in release of non-transcribing polymerases after collisions. (B) The calculated N mRNA amounts within one hour of simulations for various sliding Dsl and dissociation Koff rates with relative binding affinity to TIS R = 500 (C) and similar simulation results as in B except with R = 1. The highlighted results at left show transcription through sliding facilitated initiation on circular templates (Dsl = 104 nm2/s and Koff = 10−5/s) with polymerase distributions showing in pie charts (Top) under transcribing and (Bottom) under non-transcribing conditions. The right panel shows transcription under high Polymerase fall off rates with Koff = 10−1/s.
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pcbi-1004004-g002: Transcription of circular genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a circular genome template, (A) shows a representation of this model with collision rules resulting in release of non-transcribing polymerases after collisions. (B) The calculated N mRNA amounts within one hour of simulations for various sliding Dsl and dissociation Koff rates with relative binding affinity to TIS R = 500 (C) and similar simulation results as in B except with R = 1. The highlighted results at left show transcription through sliding facilitated initiation on circular templates (Dsl = 104 nm2/s and Koff = 10−5/s) with polymerase distributions showing in pie charts (Top) under transcribing and (Bottom) under non-transcribing conditions. The right panel shows transcription under high Polymerase fall off rates with Koff = 10−1/s.

Mentions: We tested the model with circular templates that included sliding of non-transcribing polymerases with a diffusion coefficient Dsl and a fall off rate of Koff as shown in Fig. 2. In this model we could recover the transcription even under low TIS binding strength (R≤10) while keeping the polymerases bound to the template when transcription stopped. Under the most favorable conditions R = 1, Dsl = 104 nm2/s Koff = 6.4×10−6/s as highlighted in Fig. 2C left panel, a total of 80.6±6.5 N mRNA molecules were synthesized in one hour of transcription. More careful investigation into the histories of the polymerases in this model revealed that contrary to the sliding facilitated polymerase release model highlighted in Fig. 1C left panel, 99.5% of polymerases found and engaged the TIS through 1D diffusion. Although free polymerases contributed only 0.5% to transcription initiation under the circular genome model, 99% of the free polymerases were still released into the solution through collision with transcribing polymerases. The presence of free polymerases during transcription combined by their tight association under non-transcribing conditions is in agreement with results showing redistribution of polymerases from UV irradiated templates [28].


Vesicular stomatitis virus polymerase's strong affinity to its template suggests exotic transcription models.

Tang X, Bendjennat M, Saffarian S - PLoS Comput. Biol. (2014)

Transcription of circular genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a circular genome template, (A) shows a representation of this model with collision rules resulting in release of non-transcribing polymerases after collisions. (B) The calculated N mRNA amounts within one hour of simulations for various sliding Dsl and dissociation Koff rates with relative binding affinity to TIS R = 500 (C) and similar simulation results as in B except with R = 1. The highlighted results at left show transcription through sliding facilitated initiation on circular templates (Dsl = 104 nm2/s and Koff = 10−5/s) with polymerase distributions showing in pie charts (Top) under transcribing and (Bottom) under non-transcribing conditions. The right panel shows transcription under high Polymerase fall off rates with Koff = 10−1/s.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263359&req=5

pcbi-1004004-g002: Transcription of circular genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a circular genome template, (A) shows a representation of this model with collision rules resulting in release of non-transcribing polymerases after collisions. (B) The calculated N mRNA amounts within one hour of simulations for various sliding Dsl and dissociation Koff rates with relative binding affinity to TIS R = 500 (C) and similar simulation results as in B except with R = 1. The highlighted results at left show transcription through sliding facilitated initiation on circular templates (Dsl = 104 nm2/s and Koff = 10−5/s) with polymerase distributions showing in pie charts (Top) under transcribing and (Bottom) under non-transcribing conditions. The right panel shows transcription under high Polymerase fall off rates with Koff = 10−1/s.
Mentions: We tested the model with circular templates that included sliding of non-transcribing polymerases with a diffusion coefficient Dsl and a fall off rate of Koff as shown in Fig. 2. In this model we could recover the transcription even under low TIS binding strength (R≤10) while keeping the polymerases bound to the template when transcription stopped. Under the most favorable conditions R = 1, Dsl = 104 nm2/s Koff = 6.4×10−6/s as highlighted in Fig. 2C left panel, a total of 80.6±6.5 N mRNA molecules were synthesized in one hour of transcription. More careful investigation into the histories of the polymerases in this model revealed that contrary to the sliding facilitated polymerase release model highlighted in Fig. 1C left panel, 99.5% of polymerases found and engaged the TIS through 1D diffusion. Although free polymerases contributed only 0.5% to transcription initiation under the circular genome model, 99% of the free polymerases were still released into the solution through collision with transcribing polymerases. The presence of free polymerases during transcription combined by their tight association under non-transcribing conditions is in agreement with results showing redistribution of polymerases from UV irradiated templates [28].

Bottom Line: Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial.We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies.In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of Utah, Salt Lake City, Utah, United States of America.

ABSTRACT
Vesicular stomatitis virus (VSV) is the prototype for negative sense non segmented (NNS) RNA viruses which include potent human and animal pathogens such as Rabies, Ebola and measles. The polymerases of NNS RNA viruses only initiate transcription at or near the 3' end of their genome template. We measured the dissociation constant of VSV polymerases from their whole genome template to be 20 pM. Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial. To explore possible mechanisms, we simulated the first hour of transcription using Monte Carlo methods and show that a one-time initial dissociation of all polymerases during entry is not sufficient to sustain transcription. We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies. In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.

Show MeSH
Related in: MedlinePlus