Road rules for traffic on DNA – systematic analysis of transcriptional roadblocking in vivo,. Hao, N., Krishna, S., Ahlgren-Berg, A., Cutts, E. E, Shearwin, K. E, & Dodd, I. B Nucl. Acids Res., 42:8861–8872, 2014. Pdf doi abstract bibtex Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining the magnitude of transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how roadblocking by the lac repressor (LacI) in Escherichia coli cells is controlled by promoter firing rate, the concentration and affinity of the roadblocker protein, the transcription-coupled repair protein Mfd, and promoter–roadblock spacing. Increased readthrough of the roadblock at higher RNAP fluxes requires active dislodgement of LacI by multiple RNAPs. However, this RNAP cooperation effect occurs only for strong promoters because roadblock-paused RNAP is quickly terminated by Mfd. The results are most consistent with a single RNAP also sometimes dislodging LacI, though we cannot exclude the possibility that a single RNAP reads through by waiting for spontaneous LacI dissociation. Reducing the occupancy of the roadblock site by increasing the LacI off-rate (weakening the operator) increased dislodgement strongly, giving a stronger effect on readthrough than decreasing the LacI on-rate (decreasing LacI concentration). Thus, protein binding kinetics can be tuned to maintain site occupation while reducing detrimental roadblocking.
@article{roadrules,
title = {Road rules for traffic on DNA -- systematic analysis of transcriptional roadblocking in vivo,},
author = {Nan Hao and Sandeep Krishna and Alexandra Ahlgren-Berg and Erin E Cutts and Keith E Shearwin and Ian B Dodd},
journal = {Nucl. Acids Res.},
year = {2014},
volume = {42},
pages = {8861--8872},
abstract = {Genomic DNA is bound by many proteins that could
potentially impede elongation of RNA polymerase
(RNAP), but the factors determining the magnitude of
transcriptional roadblocking in vivo are poorly understood.
Through systematic experiments and modeling,
we analyse how roadblocking by the lac repressor
(LacI) in Escherichia coli cells is controlled by
promoter firing rate, the concentration and affinity
of the roadblocker protein, the transcription-coupled
repair protein Mfd, and promoter–roadblock spacing.
Increased readthrough of the roadblock at higher
RNAP fluxes requires active dislodgement of LacI
by multiple RNAPs. However, this RNAP cooperation
effect occurs only for strong promoters because
roadblock-paused RNAP is quickly terminated by
Mfd. The results are most consistent with a single
RNAP also sometimes dislodging LacI, though we
cannot exclude the possibility that a single RNAP
reads through by waiting for spontaneous LacI dissociation.
Reducing the occupancy of the roadblock
site by increasing the LacI off-rate (weakening the
operator) increased dislodgement strongly, giving a
stronger effect on readthrough than decreasing the
LacI on-rate (decreasing LacI concentration). Thus,
protein binding kinetics can be tuned to maintain site
occupation while reducing detrimental roadblocking.},
keywords={Bacterial genetics,Biological physics,Gene regulation,Genetic circuit engineering},
url_PDf={Hao_2014_NuclAcidsRes_Roadblocking.pdf},
doi={10.1093/nar/gku627}
}
Downloads: 0
{"_id":"icMHZeXJXYXWuY9PJ","bibbaseid":"hao-krishna-ahlgrenberg-cutts-shearwin-dodd-roadrulesfortrafficondnasystematicanalysisoftranscriptionalroadblockinginvivo-2014","author_short":["Hao, N.","Krishna, S.","Ahlgren-Berg, A.","Cutts, E. E","Shearwin, K. E","Dodd, I. B"],"bibdata":{"bibtype":"article","type":"article","title":"Road rules for traffic on DNA – systematic analysis of transcriptional roadblocking in vivo,","author":[{"firstnames":["Nan"],"propositions":[],"lastnames":["Hao"],"suffixes":[]},{"firstnames":["Sandeep"],"propositions":[],"lastnames":["Krishna"],"suffixes":[]},{"firstnames":["Alexandra"],"propositions":[],"lastnames":["Ahlgren-Berg"],"suffixes":[]},{"firstnames":["Erin","E"],"propositions":[],"lastnames":["Cutts"],"suffixes":[]},{"firstnames":["Keith","E"],"propositions":[],"lastnames":["Shearwin"],"suffixes":[]},{"firstnames":["Ian","B"],"propositions":[],"lastnames":["Dodd"],"suffixes":[]}],"journal":"Nucl. Acids Res.","year":"2014","volume":"42","pages":"8861–8872","abstract":"Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining the magnitude of transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how roadblocking by the lac repressor (LacI) in Escherichia coli cells is controlled by promoter firing rate, the concentration and affinity of the roadblocker protein, the transcription-coupled repair protein Mfd, and promoter–roadblock spacing. Increased readthrough of the roadblock at higher RNAP fluxes requires active dislodgement of LacI by multiple RNAPs. However, this RNAP cooperation effect occurs only for strong promoters because roadblock-paused RNAP is quickly terminated by Mfd. The results are most consistent with a single RNAP also sometimes dislodging LacI, though we cannot exclude the possibility that a single RNAP reads through by waiting for spontaneous LacI dissociation. Reducing the occupancy of the roadblock site by increasing the LacI off-rate (weakening the operator) increased dislodgement strongly, giving a stronger effect on readthrough than decreasing the LacI on-rate (decreasing LacI concentration). Thus, protein binding kinetics can be tuned to maintain site occupation while reducing detrimental roadblocking.","keywords":"Bacterial genetics,Biological physics,Gene regulation,Genetic circuit engineering","url_pdf":"Hao_2014_NuclAcidsRes_Roadblocking.pdf","doi":"10.1093/nar/gku627","bibtex":"@article{roadrules,\r\n title = {Road rules for traffic on DNA -- systematic analysis of transcriptional roadblocking in vivo,},\r\n author = {Nan Hao and Sandeep Krishna and Alexandra Ahlgren-Berg and Erin E Cutts and Keith E Shearwin and Ian B Dodd},\r\n journal = {Nucl. Acids Res.},\r\n year = {2014},\r\n volume = {42},\r\n pages = {8861--8872},\r\n abstract = {Genomic DNA is bound by many proteins that could\r\npotentially impede elongation of RNA polymerase\r\n(RNAP), but the factors determining the magnitude of\r\ntranscriptional roadblocking in vivo are poorly understood.\r\nThrough systematic experiments and modeling,\r\nwe analyse how roadblocking by the lac repressor\r\n(LacI) in Escherichia coli cells is controlled by\r\npromoter firing rate, the concentration and affinity\r\nof the roadblocker protein, the transcription-coupled\r\nrepair protein Mfd, and promoter–roadblock spacing.\r\nIncreased readthrough of the roadblock at higher\r\nRNAP fluxes requires active dislodgement of LacI\r\nby multiple RNAPs. However, this RNAP cooperation\r\neffect occurs only for strong promoters because\r\nroadblock-paused RNAP is quickly terminated by\r\nMfd. The results are most consistent with a single\r\nRNAP also sometimes dislodging LacI, though we\r\ncannot exclude the possibility that a single RNAP\r\nreads through by waiting for spontaneous LacI dissociation.\r\nReducing the occupancy of the roadblock\r\nsite by increasing the LacI off-rate (weakening the\r\noperator) increased dislodgement strongly, giving a\r\nstronger effect on readthrough than decreasing the\r\nLacI on-rate (decreasing LacI concentration). Thus,\r\nprotein binding kinetics can be tuned to maintain site\r\noccupation while reducing detrimental roadblocking.},\r\n keywords={Bacterial genetics,Biological physics,Gene regulation,Genetic circuit engineering},\r\n url_PDf={Hao_2014_NuclAcidsRes_Roadblocking.pdf},\r\n doi={10.1093/nar/gku627}\r\n}\r\n","author_short":["Hao, N.","Krishna, S.","Ahlgren-Berg, A.","Cutts, E. E","Shearwin, K. E","Dodd, I. B"],"key":"roadrules","id":"roadrules","bibbaseid":"hao-krishna-ahlgrenberg-cutts-shearwin-dodd-roadrulesfortrafficondnasystematicanalysisoftranscriptionalroadblockinginvivo-2014","role":"author","urls":{" pdf":"https://theory.ncbs.res.in/sandeep/publications/Hao_2014_NuclAcidsRes_Roadblocking.pdf"},"keyword":["Bacterial genetics","Biological physics","Gene regulation","Genetic circuit engineering"],"metadata":{"authorlinks":{}},"html":""},"bibtype":"article","biburl":"https://theory.ncbs.res.in/sandeep/publications/sandeeppublications.bib","dataSources":["Kf28CgddoYx9JQDzp"],"keywords":["bacterial genetics","biological physics","gene regulation","genetic circuit engineering"],"search_terms":["road","rules","traffic","dna","systematic","analysis","transcriptional","roadblocking","vivo","hao","krishna","ahlgren-berg","cutts","shearwin","dodd"],"title":"Road rules for traffic on DNA – systematic analysis of transcriptional roadblocking in vivo,","year":2014}