Peer Review #2 of "In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli (v0.1)"

A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor-operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

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Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.19.6708-6718.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6708-6718 ◽

Cited By ~ 17

Author(s):

Jason R. Wickstrum ◽

Thomas J. Santangelo ◽

Susan M. Egan

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

In Vitro Transcription ◽

Dnase I ◽

Receptor Protein ◽

Transcription Activator ◽

Transcription Start ◽

Dnase I Footprinting

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

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Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

Journal of Bacteriology ◽

10.1128/jb.185.9.2920-2926.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2920-2926 ◽

Cited By ~ 17

Author(s):

Wilson B. Muse ◽

Christopher J. Rosario ◽

Robert A. Bender

Keyword(s):

Escherichia Coli ◽

Nitrogen Assimilation ◽

Cytosine Deaminase ◽

In Vitro Transcription ◽

Dependent Manner ◽

E Coli ◽

Different Response ◽

Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

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Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region

Journal of Bacteriology ◽

10.1128/jb.152.1.363-371.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 363-371

Author(s):

S P Lynn ◽

J F Gardner ◽

W S Reznikoff

Keyword(s):

Escherichia Coli ◽

Transcription Termination ◽

Regulatory Region ◽

In Vitro Transcription ◽

Regulatory Mutation ◽

Rna Transcripts ◽

In Vitro Transcripts ◽

K 12

Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.

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In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

10.7287/peerj.preprints.317v1 ◽

2014 ◽

Author(s):

Matthew Sochor

Keyword(s):

Transcriptional Repression ◽

In Vitro Transcription ◽

Lac Repressor ◽

Molecular Crowding ◽

Binding Data ◽

Direct Modeling ◽

Operator Dna ◽

The Given

A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor-operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

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An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.10.2740-2747.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2740-2747 ◽

Cited By ~ 30

Author(s):

Sabine Brantl ◽

E. Gerhart H. Wagner

Keyword(s):

Escherichia Coli ◽

Antisense Rna ◽

In Vitro Transcription ◽

Control Mode ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Antisense Rnas ◽

Replication Control

ABSTRACT Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

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