DOI: 10.5061/dryad.jm63xsjgt
title: Transcription attenuation in synthetic promoters in nonoverlapping tandem formation

description: 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.
description: This READ ME.md file was generated on 10-04-2024. # Transcription
 Attenuation in Synthetic Promoters in Nonoverlapping Tandem Formation ---
 ### 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. - 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). - 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. - 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. - 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. - 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. - 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. - 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. ##### 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