Local genetic context shapes the function of a gene regulatory network
- Institute of Science and Technology Austria, Austria
- Immunobiology and Genetics, Max F. Perutz Laboratories, Center Of Molecular Biology, University of Vienna, Austria
Figures
Figure 1 with 5 supplements
Architecture and phenotypes of the gene regulatory network (GRN).
(A) Diagram of interactions between the three independent transcriptional units (TUs) encoding for the repressors, lacI, tetR, and cI, their respective inducers isopropyl β-D-thiogalactopyranoside …
Figure 1—figure supplement 1
A model of the mechanistic basis of gene expression, including transcriptional read-through.
(A) The topology of network interactions used in the model, along with the equations that model them. Term in red represents transcriptional read-through. (B) Two examples of transcriptional …
Figure 1—figure supplement 2
Gene regulatory networks (GRNs) in which phenotype is dependent only on changes in relative transcriptional unit (TU) order and orientation.
Fluorescence of cells carrying 26 different TU permutations of the GRN plasmid, grouped in pairs differing only in orientation with respect to plasmid backbone. Graphs show means and error bars …
Figure 1—figure supplement 3
Gene regulatory networks (GRNs) in which the influence of plasmid-encoded genetic elements cannot be ruled out.
Fluorescence of cells carrying 11 different transcriptional unit (TU) permutations of the GRN plasmid, grouped in pairs differing only in orientation with respect to plasmid backbone. Shading marks …
Figure 1—figure supplement 4
Influence of plasmid genetic elements on gene expression levels.
(A) Fluorescence of cells carrying a promoterless yfp gene cloned adjacent to the origin of replication, ori. (B) Fluorescence of cells carrying Ptet-yfp cloned in two orientations relative to ori …
Figure 1—figure supplement 5
Distribution of phenotypes depending on the threshold applied to define ON and OFF states.
(A) Definition of the logic operations. The presence or absence of inducer is indicated with + and −, and output by On and Off. (B) Histogram shows the fraction of networks that can be assigned to a …
Figure 2
Changes in relative transcriptional unit (TU) order lead to qualitative changes of phenotype.
Fluorescence of cells carrying six different TU permutations of the gene regulatory network (GRN) on a plasmid (A), three GRN variants integrated on the chromosome at the phage HK022 attachment site …
Figure 3
Differences in cI expression lead to transcriptional unit (TU) order dependent phenotypes.
(A) RT-qPCR analysis of cI expression. RT-qPCR was performed, using cI-specific primers. The induction ratios were calculated relative to the uninduced strain CLT. (B) YFP levels measured in strains …
Figure 4 with 2 supplements
Phenotype of strain TLC can be explained by transcriptional read-through.
(A) Genetic architecture of plasmid fragments encoding three repressors in strains TLC and TLC-10 (carrying mutations in the −10 promoter element of Ptet). Promoters are marked as bent arrows, …
Figure 4—figure supplement 1
Phenotype of strains LCT, TrCrLr, TCL, LrCrTr, TLC, and CrLrTr can be explained by transcriptional read-through.
Genetic architecture of plasmid fragments encoding three repressors in strains LCT and TrCrLr (A), TCL and LrCrTr (B), TLC and CrLrTr (C), and their derivatives carrying mutations in the −10 …
Figure 4—figure supplement 2
Point mutations in −10 promoter element render Ptet inactive.
Fluorescence of cells carrying either wild-type Ptet or Ptet with two point mutations in the −10 element driving yfp expression. Strains were grown without inducers. Graphs show means and error bars …
Figure 5 with 2 supplements
Change of terminator leads to qualitative change in phenotype.
(A) Genetic architecture of plasmid fragments encoding three repressors in strains TLC with different terminators: T1, T1T2, Tcrp, or TtonB, preceding cI, and fluorescence of cells carrying these …
Figure 5—figure supplement 1
Northern blot assay shows read-through transcript in strains carrying Tcrp and TtonB.
(A) Schematic representation of the plasmid fragments encoding three repressors in strains TLC, TLTcrpC, and TLTtonBC. Shading marks regions which differ between the three gene regulatory networks (G…
Figure 5—figure supplement 2
Terminator containing DNA fragments between repressor genes show no cryptic promoter activity.
Fluorescence of cells carrying DNA fragments located on gene regulatory network (GRN) plasmids between lacI and cI, and encoding T1, Tcrp, and TtonB, cloned in front of a promoterless yfp gene, and …
Figure 6 with 1 supplement
Effect of genetic context of lacI repressor chromosomal position on Plac activity.
(A) Diagram of interactions between LacI repressor and the promoter it represses, as well as genetic architecture of the DNA fragments integrated into MG1655 ΔlacI ΔlacZYA strain. Promoters are …
Figure 6—figure supplement 1
Plac activity differs significantly in cells carrying lacI inserted after a weak (yeaH), medium (flhC), and strong (asnT) terminator grown in different concentrations of isopropyl β-D-thiogalactopyranoside (IPTG).
Bars are mean values, circles individual measurements. Error bars are standard deviations. Stars indicate strains that significantly differ from each other (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0…
Figure 7
Architecture of a regulon depends on local genetic context.
(A) Diagram of interactions within our gene regulatory network (GRN) in two different transcriptional unit (TU) arrangements: CLT and TLC. (B) Regulatory patterns in two regulons with overlapping …
Additional files
-
Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/65993/elife-65993-supp1-v3.docx
-
Supplementary file 2
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/65993/elife-65993-supp2-v3.docx
-
Supplementary file 3
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/65993/elife-65993-supp3-v3.docx
-
Supplementary file 4
ANOVA test statistics.
- https://cdn.elifesciences.org/articles/65993/elife-65993-supp4-v3.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/65993/elife-65993-transrepform-v3.docx
