Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli

Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are u...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Yan, Qiang [verfasserIn] Fong, Stephen S.

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2017

Schlagwörter:	Upstream element sequence RNAP α subunits Gene expression noise Promoter strength RNA polymerase Binding association constant

Anmerkung:	© The Author(s). 2017

Übergeordnetes Werk:	Enthalten in: Journal of biological engineering - Berlin : Springer, 2007, 11(2017), 1 vom: 01. Nov.
Übergeordnetes Werk:	volume:11 ; year:2017 ; number:1 ; day:01 ; month:11

Links:	Volltext

DOI / URN:	10.1186/s13036-017-0075-2

Katalog-ID:	SPR029569028

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520			\|a Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes.
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allfields	10.1186/s13036-017-0075-2 doi (DE-627)SPR029569028 (SPR)s13036-017-0075-2-e DE-627 ger DE-627 rakwb eng Yan, Qiang verfasserin (orcid)0000-0002-5038-5776 aut Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213 Fong, Stephen S. aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 11(2017), 1 vom: 01. Nov. (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:11 year:2017 number:1 day:01 month:11 https://dx.doi.org/10.1186/s13036-017-0075-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2017 1 01 11
spelling	10.1186/s13036-017-0075-2 doi (DE-627)SPR029569028 (SPR)s13036-017-0075-2-e DE-627 ger DE-627 rakwb eng Yan, Qiang verfasserin (orcid)0000-0002-5038-5776 aut Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213 Fong, Stephen S. aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 11(2017), 1 vom: 01. Nov. (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:11 year:2017 number:1 day:01 month:11 https://dx.doi.org/10.1186/s13036-017-0075-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2017 1 01 11
allfields_unstemmed	10.1186/s13036-017-0075-2 doi (DE-627)SPR029569028 (SPR)s13036-017-0075-2-e DE-627 ger DE-627 rakwb eng Yan, Qiang verfasserin (orcid)0000-0002-5038-5776 aut Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213 Fong, Stephen S. aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 11(2017), 1 vom: 01. Nov. (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:11 year:2017 number:1 day:01 month:11 https://dx.doi.org/10.1186/s13036-017-0075-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2017 1 01 11
allfieldsGer	10.1186/s13036-017-0075-2 doi (DE-627)SPR029569028 (SPR)s13036-017-0075-2-e DE-627 ger DE-627 rakwb eng Yan, Qiang verfasserin (orcid)0000-0002-5038-5776 aut Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213 Fong, Stephen S. aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 11(2017), 1 vom: 01. Nov. (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:11 year:2017 number:1 day:01 month:11 https://dx.doi.org/10.1186/s13036-017-0075-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2017 1 01 11
allfieldsSound	10.1186/s13036-017-0075-2 doi (DE-627)SPR029569028 (SPR)s13036-017-0075-2-e DE-627 ger DE-627 rakwb eng Yan, Qiang verfasserin (orcid)0000-0002-5038-5776 aut Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213 Fong, Stephen S. aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 11(2017), 1 vom: 01. Nov. (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:11 year:2017 number:1 day:01 month:11 https://dx.doi.org/10.1186/s13036-017-0075-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2017 1 01 11
language	English
source	Enthalten in Journal of biological engineering 11(2017), 1 vom: 01. Nov. volume:11 year:2017 number:1 day:01 month:11
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topic_facet	Upstream element sequence RNAP α subunits Gene expression noise Promoter strength RNA polymerase Binding association constant
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author	Yan, Qiang
spellingShingle	Yan, Qiang misc Upstream element sequence misc RNAP α subunits misc Gene expression noise misc Promoter strength misc RNA polymerase misc Binding association constant Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli
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topic_title	Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli Upstream element sequence (dpeaa)DE-He213 RNAP α subunits (dpeaa)DE-He213 Gene expression noise (dpeaa)DE-He213 Promoter strength (dpeaa)DE-He213 RNA polymerase (dpeaa)DE-He213 Binding association constant (dpeaa)DE-He213
topic	misc Upstream element sequence misc RNAP α subunits misc Gene expression noise misc Promoter strength misc RNA polymerase misc Binding association constant
topic_unstemmed	misc Upstream element sequence misc RNAP α subunits misc Gene expression noise misc Promoter strength misc RNA polymerase misc Binding association constant
topic_browse	misc Upstream element sequence misc RNAP α subunits misc Gene expression noise misc Promoter strength misc RNA polymerase misc Binding association constant
format_facet	Elektronische Aufsätze Aufsätze Elektronische Ressource
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title	Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli
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title_full	Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli
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author_browse	Yan, Qiang Fong, Stephen S.
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title_sort	study of in vitro transcriptional binding effects and noise using constitutive promoters combined with up element sequences in escherichia coli
title_auth	Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli
abstract	Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. © The Author(s). 2017
abstractGer	Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. © The Author(s). 2017
abstract_unstemmed	Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants ($ K_{d} $) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < $ K_{d} $ < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes. © The Author(s). 2017
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700
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title_short	Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli
url	https://dx.doi.org/10.1186/s13036-017-0075-2
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Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli

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