Transcription attenuation in synthetic promoters in nonoverlapping tandem formation
Abstract
Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters’ positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.
README: Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation
This READ ME.md file was generated on 10-04-2024.
1. Principal Investigator contact information:
- Name: Andre S. Ribeiro
- Institution: Tampere University
- Email: andre.sanchesribeiro@tuni.fi
2. Description of the dataset:
This dataset contains flow cytometry and spectrophotometry data reagarding synthetic cosntructs of individual promoters (PLacO3O1, PtetA and PBAD) and of those promoters when in tandem formation (PLacO3O1-PtetA, PtetA-PLacO3O1 and PtetA-PBAD-PLacO3O1).
Description of the data and file structure
1. File list:
Flow Cytometry: This folder contains all flow cytometry data used in the project.
Note: Unless stated otherwise, in the subfolders, there are 3 CSV files labelled "Sample 1", "Sample 2", and "Sample 3" that represent the measurements of 3 biological replicates in a given condition.
01 Single point measurements: Contains data measured by flow cytometry for a single condition.
- 01 Control: Flow cytometry data related to measurements of DH5α-Pro cells without synthetic plasmids.
- 01 WT: Measurements of DH5α-Pro cells.
- 02 WT + aTc: Measurements of DH5α-Pro cells with the inducer aTc.
02 Individual promoters: Flow cytometry data related to measurements of individual promoters.
- 01 PLacO3O1: Flow cytometry data related to the individual promoter PLacO3O1.
- 01 Uninduced: Measurements when the promoter is not induced.
- 02 Induced: Measurements when the promoter is fully induced.
- 03-06 Rifampicin X ug_mL: Measurements when the fully induced promoter is subjected to X μg/mL of the antibiotic rifampicin.
- 02 PtetA: Flow cytometry data related to the individual promoter PtetA.
- 01 Uninduced: Measurements when the promoter is not induced.
- 02 Induced: Measurements when the promoter is fully induced.
- 03-06 Rifampicin X ug_mL: Measurements when the fully induced promoter is subjected to X μg/mL of the antibiotic rifampicin.
- 03 PBAD: Flow cytometry data related to the individual promoter PBAD.
- 01 Uninduced: Measurements when the promoter is not induced.
- 02 Induced: Measurements when the promoter is fully induced.
- 03-04 Rifampicin X ug_mL: Measurements when the fully induced promoter is subjected to X μg/mL of the antibiotic rifampicin.
- 01 PLacO3O1: Flow cytometry data related to the individual promoter PLacO3O1.
03 Tandem promoters: Flow cytometry data related to measurements of tandem promoters.
01 150 bp: Flow cytometry data related to measurements of tandem promoters whose inter-promoter spacing (between TSS positions) is 150 bp.
- 01 PtetA-PLacO3O1: Flow cytometry data related to the tandem construct PtetA-PLacO3O1.
- 01 Uninduced: Measurements when none of the tandem promoters are induced.
- 02 PLacO3O1 Induced: Measurements when only the promoter PLacO3O1 is fully induced.
- 03 PtetA Induced: Measurements when only the promoter PtetA is fully induced.
- 04 Both Induced: Measurements when both promoters are fully induced.
- 05-08 Rifampicin X ug_mL: Measurements when the construct is subjected to X μg/mL of the antibiotic rifampicin (with both promoters fully induced).
- 02 PLacO3O1-PtetA: Flow cytometry data related to the tandem construct PLacO3O1-PtetA.
- 01 Uninduced: Measurements when none of the tandem promoters are induced.
- 02 PLacO3O1 Induced: Measurements when only the promoter PLacO3O1 is fully induced.
- 03 PtetA Induced: Measurements when only the promoter PtetA is fully induced.
- 04 Both Induced: Measurements when both promoters are fully induced.
- 05-08 Rifampicin X ug_mL: Measurements when the construct is subjected to X μg/mL of the antibiotic rifampicin (with both promoters fully induced).
- 03 PtetA-PBAD-PLacO3O1: Flow cytometry data related to the tandem construct PtetA-PBAD-PLacO3O1.
- 01 Uninduced: Measurements when none of the tandem promoters are induced.
- 02 PLacO3O1 Induced: Measurements when only the promoter PLacO3O1 is fully induced.
- 03 PtetA Induced: Measurements when only the promoter PtetA is fully induced.
- 04 PBAD Induced: Measurements when only the promoter PBAD is fully induced.
- 05 All Induced: Measurements when all promoters are fully induced.
- 05-07 Rifampicin X ug_mL: Measurements when the construct is subjected to X μg/mL of the antibiotic rifampicin (with all promoters fully induced).
- 01 PtetA-PLacO3O1: Flow cytometry data related to the tandem construct PtetA-PLacO3O1.
02 250 bp: Flow cytometry data related to measurements of tandem promoters whose inter-promoter spacing (between TSS positions) is 250 bp.
- 01 PLacO3O1-PtetA: Flow cytometry data related to the tandem construct PLacO3O1-PtetA.
- 01 Both Induced: Measurements when both promoters are fully induced. Note: The 3 CSV files labelled "Sample 1", "Sample 2", and "Sample 3" represent the measurements of 3 technical replicates.
- 01 PLacO3O1-PtetA: Flow cytometry data related to the tandem construct PLacO3O1-PtetA.
03 350 bp: Flow cytometry data related to measurements of tandem promoters whose inter-promoter spacing (between TSS positions) is 350 bp.
- 01 PtetA-PLacO3O1: Flow cytometry data related to the tandem construct PtetA-PLacO3O1.
- 01 Both Induced: Measurements when both promoters are fully induced. Note: The 3 CSV files labelled "Sample 1", "Sample 2", and "Sample 3" represent the measurements of 3 technical replicates.
- 01 PtetA-PLacO3O1: Flow cytometry data related to the tandem construct PtetA-PLacO3O1.
- 01 Control: Flow cytometry data related to measurements of DH5α-Pro cells without synthetic plasmids.
02 Induction curves: Contains data measured by flow cytometry for measurement when the inducer levels where gradually increased.
01 Individual promoters: Flow cytometry data related to induction measurements of of individual promoters.
- 01 PLacO3O1 Induction: Flow cytometry data related to the gradual induction of the promoter PLacO3O1 with IPTG.
- 01-08 IPTG X uM: Measurements when the promoter is induced with X μM of IPTG.
- 01-08 IPTG X uM: Measurements when the promoter is induced with X μM of IPTG.
- 02 PtetA Induction: Flow cytometry data related to the gradual induction of the promoter PtetA with aTc. - 01-08 aTc X uM: Measurements when the promoter is induced with X ng/mL of aTc.
- 03 PBAD Induction: Flow cytometry data related to the gradual induction of the promoter PBAD with Arabinose. - 01-07 Arabinose X %: Measurements when the promoter is induced with X % of Arabinose.
- 01 PLacO3O1 Induction: Flow cytometry data related to the gradual induction of the promoter PLacO3O1 with IPTG.
02 Tandem promoters: Flow cytometry data related to induction measurements of tandem promoters.
01 PtetA-PLacO3O1: Flow cytometry data related to the gradual induction of the tandem construct PtetA-PLacO3O1.
- 01 PLacO3O1 Induction: Flow cytometry data related to the gradual induction of the downstream promoter PLacO3O1 with IPTG. The upstream promoter PtetA is fully induced.
- 01-08 IPTG X uM: Measurements when the downstream promoter PLacO3O1 is induced with X μM of IPTG.
- 01-08 IPTG X uM: Measurements when the downstream promoter PLacO3O1 is induced with X μM of IPTG.
- 02 PtetA Induction: Flow cytometry data related to the gradual induction of the upstream promoter PtetA with IPTG. The downstream promoter PLacO3O1 is fully induced.
- 01-08 aTc X ng/mL: Measurements when the upstream promoter PtetA is induced with X ng/mL of aTc.
- 01 PLacO3O1 Induction: Flow cytometry data related to the gradual induction of the downstream promoter PLacO3O1 with IPTG. The upstream promoter PtetA is fully induced.
Spectrophotometry: This folder contains all spectrophotometry data used in the project.
01 Growth: This folder contains an excel file (named "OD600 data") with the growth measurements for all strains used in the study and for rifampicin conditions.
Each condition contains 3 columns accounting for the 3 biological replicates measured.02 Fluorescence: This folder contains an excel file (name "Fluoresence") with the fluorescence levels for WT cells and the synthetic constructs PtetA, PLacO3O1, PtetA-PLacO3O1, and PLacO3O1-PtetA.
Each construct was measured when uninduced, when induced, and when sujected to 2.5 μg/mL and 5.0 μg/mL of the antibiotic rifampicin.
WT cells (i.e, cells without the synthetic plasmids) were measured without rifampicin and with 2.5 μg/mL and 5.0 μg/mL of rifampicin.
Each condition contains 3 columns accounting for the 3 biological replicates measured.
Plasmid Sequences: This folder contains the plasmid maps (generated by Snapgene) and FASTA files of their bioparts
01 FASTA files: Contains FA files with the sequences of the bioparts of the synthetic constructs.
- 01 Individual promoters: Sequence for the individual promoter PLacO3O1, the individual promoter PtetA, and the individual promoter PBAD.
- 02 Tandem promoters:
- 01 PtetA-PLacO3O1: Sequence for the tandem constructs PtetA-PLacO3O1. -01 150 bp: Sequence for the construct in which the tandem promoters inter-promoter spacing (between TSS positions) is 150 bp. -02 350 bp: Sequence for the construct in which the tandem promoters inter-promoter spacing (between TSS positions) is 350 bp.
- 02 PLacO3O1-PtetA: Sequence for the tandem constructs PLacO3O1-PtetA. -01 150 bp: Sequence for the construct in which the tandem promoters inter-promoter spacing (between TSS positions) is 150 bp. -02 250 bp: Sequence for the construct in which the tandem promoters inter-promoter spacing (between TSS positions) is 250 bp.
- 03 PtetA-PBAD-PLacO3O1: Sequence for the tandem construct PtetA-PBAD-PLacO3O1.
"Plasmid maps" word file: Contains the plasmid maps (generated by Snapgene) of the synthetic plasmids used in the project.
2. Are there multiple versions of the dataset?
No
3. Methodical information
Description of methods used for collection/generation of data:
Collection and data generation are described in "Materials and Methods" section of main manuscript.Methods for processing the data:
Data processing methods and the software packages used are described in "Materials and Methods" section of main manuscript.
Sharing/Access information
1. Licenses placed on the data:
This work is licensed under a CC0 1.0 Universal (CC0 1.0) Public Domain Dedication license. All data in Dryad is released into the public domain under the terms of a Creative Commons Zero (CC0) waiver.
CC0 does not exempt those who reuse the data from the following community norms for scholarly communication. It does not exempt researchers from reusing the data in a way that is mindful of its limitations.
Nor does it exempt researchers from the obligation of citing the original data authors. CC0 facilitates the discovery, re-use, and citation of that data.
For more information see (https://creativecommons.org/share-your-work/public-domain/cc0).
2. Was data derived from another source?
No
3. Recommended citation for this dataset:
V. Chauhan, I.S.C. Baptista, A.M. Arsh, R. Jagadeesan, S. Dash, and A.S. Ribeiro (2024) Transcription Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation. Biochemistry. DOI: 10.1021/acs.biochem.4c00012