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Enhancement of LacI binding in vivo.

ABSTRACT: Transcription factors (TFs) bind to specific sequences in DNA to regulate transcription. Despite extensive measurements of TFs' dissociation constant (Kd) in vitro, their apparent Kdin vivo are usually unknown. LacI, a bacterial TF, is often used to artificially recruit proteins onto eukaryotic genomes. As LacI binds tightly to its recognition site (LacO) in vitro with a Kd about 10 picomolar (pM), it is often assumed that LacI also has high affinity to LacO in vivo. In this work, we measured LacI binding in living yeast cells using a fluorescent repressor operator system and found an apparent Kd of ∼0.6 μM, four orders of magnitude higher than that in vitro. By genetically altering (i) GFP-LacI structure, (ii) GFP-LacI stability, (iii) chromosome accessibility and (iv) LacO sequence, we reduced the apparent Kd to <10 nM. It turns out that the GFP tagging location and the fusion protein stability have a large effect on LacI binding, but surprisingly, chromosome accessibility only plays a mild role. These findings contribute to our quantitative understanding of the features that affect the apparent Kd of TF in cells. They also provide guidance for future design of more specific chromosomal recruitment through high-affinity TFs.

SUBMITTER: Du M

PROVIDER: S-EPMC6765135 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Enhancement of LacI binding in vivo.

Du Manyu M Kodner Seth S Bai Lu L

Nucleic acids research 20191001 18

Transcription factors (TFs) bind to specific sequences in DNA to regulate transcription. Despite extensive measurements of TFs' dissociation constant (Kd) in vitro, their apparent Kdin vivo are usually unknown. LacI, a bacterial TF, is often used to artificially recruit proteins onto eukaryotic genomes. As LacI binds tightly to its recognition site (LacO) in vitro with a Kd about 10 picomolar (pM), it is often assumed that LacI also has high affinity to LacO in vivo. In this work, we measured La ...[more]

PMID: 31396617

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Project description:Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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Enhancement of LacI binding in vivo.

Publications

Enhancement of LacI binding in vivo.

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