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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 linear genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a linear 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 TIS binding affinity R = 1. (C) Similar simulation results as in B except with R = 500. The highlighted results at left show transcription under sliding facilitated polymerase release (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-g001: Transcription of linear genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a linear 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 TIS binding affinity R = 1. (C) Similar simulation results as in B except with R = 500. The highlighted results at left show transcription under sliding facilitated polymerase release (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 simulated transcription from a set of 50 polymerases on the linear genome template using the Monte Carlo rules explained in the methods. In brief, 50 polymerases were followed on the template. Polymerases would only initiate transcription from the TIS at the 3′ end. During transcription, polymerases had a 30% probability of falling off the template at the end of each gene. Non-transcribing polymerases would slide along the template with the 1D diffusion rate Dsl and would fall off spontaneously from the genome template with the rate Koff. In the event of a collision between a transcribing and a non-transcribing polymerase, the non-transcribing polymerase would be forced off the template. Free polymerases in solution would find the template through a diffusion limited reaction and would have a binding affinity R to the TIS relative to a random binding site on the template. Neither Koff, Dsl nor R values are known experimentally, therefore we performed 4,000 simulations spanning a large range of these parameters (Dsl: 10 nm2/s to 105 nm2/s; Koff: 10−5/s to 10−1/s; R: 1 to 500). We report the measured N mRNA production for each condition as shown in Fig. 1. Initially, polymerases were positioned at the templates 5′ end. The asymmetric initial distribution was compared with random initial distribution which resulted in similar N mRNA productions as shown in S3 Figure. The most critical parameter in this model is the TIS binding strength R; we could not achieve sustained transcription with R = 1 as shown in Fig. 1A. When R was increased to 500, there were two distinct conditions that allowed sustained transcription.


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 linear genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a linear 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 TIS binding affinity R = 1. (C) Similar simulation results as in B except with R = 500. The highlighted results at left show transcription under sliding facilitated polymerase release (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-g001: Transcription of linear genome with colliding polymerases.Monte Carlo simulations of 50 polymerases on a linear 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 TIS binding affinity R = 1. (C) Similar simulation results as in B except with R = 500. The highlighted results at left show transcription under sliding facilitated polymerase release (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 simulated transcription from a set of 50 polymerases on the linear genome template using the Monte Carlo rules explained in the methods. In brief, 50 polymerases were followed on the template. Polymerases would only initiate transcription from the TIS at the 3′ end. During transcription, polymerases had a 30% probability of falling off the template at the end of each gene. Non-transcribing polymerases would slide along the template with the 1D diffusion rate Dsl and would fall off spontaneously from the genome template with the rate Koff. In the event of a collision between a transcribing and a non-transcribing polymerase, the non-transcribing polymerase would be forced off the template. Free polymerases in solution would find the template through a diffusion limited reaction and would have a binding affinity R to the TIS relative to a random binding site on the template. Neither Koff, Dsl nor R values are known experimentally, therefore we performed 4,000 simulations spanning a large range of these parameters (Dsl: 10 nm2/s to 105 nm2/s; Koff: 10−5/s to 10−1/s; R: 1 to 500). We report the measured N mRNA production for each condition as shown in Fig. 1. Initially, polymerases were positioned at the templates 5′ end. The asymmetric initial distribution was compared with random initial distribution which resulted in similar N mRNA productions as shown in S3 Figure. The most critical parameter in this model is the TIS binding strength R; we could not achieve sustained transcription with R = 1 as shown in Fig. 1A. When R was increased to 500, there were two distinct conditions that allowed sustained transcription.

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