Figures and data in Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

Version of Record: January 25, 2018
Accepted Manuscript: December 15, 2017
Accepted Manuscript: December 14, 2017

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Joseph Robert Piechura

Kapil Amarnath

Erin K O'Shea

(2017)
Natural changes in light interact with circadian regulation at promoters to control gene expression in cyanobacteria

eLife 6:e32032.

https://doi.org/10.7554/eLife.32032
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Figures
Tables
Additional files

8 figures, 6 tables and 1 additional file

Figures

Figure 1

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The circadian and light response pathways in cyanobacteria.

(A) Schematic of gene expression output of the circadian clock under Constant Light conditions. Under Constant Light intensity (dashed navy blue line), dawn gene expression (dashed maroon line) and …
https://doi.org/10.7554/eLife.32032.003

Figure 2 with 4 supplements

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Natural clear day conditions sharpen the expression of dusk genes to peak just before expected darkness.

(A) Experimental setup for testing the effects of Clear Day conditions on circadian gene expression. The upper plot shows the light intensity profiles of Low Light (black) and Clear Day (magenta) …
https://doi.org/10.7554/eLife.32032.004

Figure 2—source data 1 Normalized gene expression in Low Light and Clear Day conditions.: https://doi.org/10.7554/eLife.32032.009
Download elife-32032-fig2-data1-v3.xlsx

Figure 2—figure supplement 1

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Pigment levels of cyanobacteria grown under Low Light or Clear Day conditions reveal adjustments in the photosynthetic apparatus to optimize growth in different light conditions.

(A) Estimation of phycocyanin and chlorophyll levels in cells grown under Low Light (black) or Clear Day (magenta) conditions for two days, measured at midday of the third light period. Phycocyanin …
https://doi.org/10.7554/eLife.32032.005

Figure 2—figure supplement 2

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Gene expression dynamics of dusk and dawn circadian genes under Constant Light conditions (data from Markson et al., 2013).

(A) Gene expression dynamics of circadian genes over 24 hr in Constant Light conditions in wildtype cells (left heat map) and over 12 hr in OX-D53E cells (*rpaA-*, *kaiBC-*, *Ptrc::rpaA(D53E)*) (middle …
https://doi.org/10.7554/eLife.32032.006

Figure 2—figure supplement 3

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Dawn gene expression increases during the early part of Clear Day relative to Low Light conditions.

(A) Gene expression dynamics of dawn genes ( $n$ =169) under Low Light (top) and Clear Day (bottom) conditions. Gene expression is quantified as the log $_{2}$ fold change from the average expression of the …
https://doi.org/10.7554/eLife.32032.007

Figure 2—figure supplement 4

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The gene expression dynamics of glycogen production and breakdown enzymes change in Clear Day conditions relative to Low Light conditions.

(A) Gene expression dynamics of the dusk gene *glgP*, encoding a key enzyme in glycogen breakdown, under Low Light (black) and Clear Day (magenta) conditions as measured by RNA sequencing (left …
https://doi.org/10.7554/eLife.32032.008

Figure 3 with 2 supplements

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Rapid changes in light intensity modulate the recruitment of RNA polymerase to dusk genes to control dusk gene expression.

(A) Light intensity profiles of Low Light (black) and High Light pulse (orange) conditions, in units of $μ$ mol photons m $^{- 2}$ s $^{- 1}$ (see Materials and methods - Calibrating light conditions for more …
https://doi.org/10.7554/eLife.32032.010

Figure 3—source data 1 Normalized gene expression in High Light pulse and Shade pulse conditions.: https://doi.org/10.7554/eLife.32032.013
Download elife-32032-fig3-data1-v3.xlsx
Figure 3—source data 2 List of RNAP peaks, gene targets, and quantification of enrichment under High Light pulse and Shade pulse conditions.: https://doi.org/10.7554/eLife.32032.014
Download elife-32032-fig3-data2-v3.xlsx

Figure 3—figure supplement 1

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Rapid changes in light intensity affect dawn gene expression in an opposite direction compared to dusk gene expression.

(A) Gene expression dynamics of dawn genes ( $n$ =169) under High Light pulse conditions. Gene expression is quantified as the log $_{2}$ fold change from the average expression of the gene over all time …
https://doi.org/10.7554/eLife.32032.011

Figure 3—figure supplement 2

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Changes in RNAP enrichment and downstream dusk gene expression after rapid changes in light intensity.

(A) Changes in enrichment of RNAP upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in target dusk gene expression (right heat map) for the 82 dusk …
https://doi.org/10.7554/eLife.32032.012

Figure 4 with 4 supplements

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Changes in environmental light intensity regulate RpaA $\sim$ P DNA binding activity and RNAP recruitment to control dusk gene expression downstream of clock regulation of RpaA.

(A) Phosphorylation dynamics of RpaA under Low Light vs High Light pulse. Relative levels of phosphorylated RpaA were measured using Phos-tag Western blotting (left y-axis) in cells grown under Low …
https://doi.org/10.7554/eLife.32032.015

Figure 4—source data 1 Quantification of relative RpaA∼P levels.: https://doi.org/10.7554/eLife.32032.020
Download elife-32032-fig4-data1-v3.xlsx
Figure 4—source data 2 List of RpaA peaks, gene targets, and quantification of enrichment under High Light pulse and Shade pulse conditions.: https://doi.org/10.7554/eLife.32032.021
Download elife-32032-fig4-data2-v3.xlsx

Figure 4—figure supplement 1

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Representative Western blots used to quantify relative levels of RpaA $\sim$ P under dynamic light conditions.

(A) Representative Western Blot used to quantify levels of RpaA $\sim$ P under Low Light and Clear Day conditions. Lysates were prepared from cells harvested from either Low Light (L) or Clear Day (C) …
https://doi.org/10.7554/eLife.32032.016

Figure 4—figure supplement 2

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Changes in RpaA enrichment and downstream dusk gene expression after rapid changes in light intensity.

(A) Changes in enrichment of RpaA upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in target dusk gene expression (right heat map) for the 56 dusk …
https://doi.org/10.7554/eLife.32032.017

Figure 4—figure supplement 3

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Changes in RpaA and RNA polymerase enrichment upstream of dusk genes after rapid changes in light intensity.

(A) Changes in enrichment of RpaA upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in RNAP enrichment upstream of the same gene (right heat map) …
https://doi.org/10.7554/eLife.32032.018

Figure 4—figure supplement 4

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Multifactorial behavior of RpaA $\sim$ P at select promoters under changes in light intensity.

(A)-(C) Normalized ChIP-seq signal of RpaA (red), RpaB (blue), RNAP (green) and mock IP (black) upstream of the (A) the representative dusk gene *Synpcc7942_2267*, (B) the *kaiBC* operon and (C) another …
https://doi.org/10.7554/eLife.32032.019

Figure 5 with 3 supplements

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Light-induced changes in RpaB $\sim$ P levels modulate RpaB and RNAP binding upstream of dusk genes to directly regulate dusk gene expression in response to light.

(A) Phosphorylation dynamics of RpaB under Low Light vs High Light pulse. Relative levels of phosphorylated RpaB were measured using Phos-tag Western blotting (left y-axis) in cells grown under Low …
https://doi.org/10.7554/eLife.32032.022

Figure 5—source data 1 Quantification of relative RpaB∼P levels.: https://doi.org/10.7554/eLife.32032.026
Download elife-32032-fig5-data1-v3.xlsx
Figure 5—source data 2 List of RpaB peaks, gene targets, and quantification of enrichment under High Light pulse and Shade pulse conditions.: https://doi.org/10.7554/eLife.32032.027
Download elife-32032-fig5-data2-v3.xlsx

Figure 5—figure supplement 1

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Representative Western blots used to quantify relative levels of RpaB $\sim$ P under dynamic light conditions.

(A) Representative Western Blot used to quantify levels of RpaB $\sim$ P under Low Light and Clear Day conditions. Lysates were prepared from cells harvested from either Low Light (L) or Clear Day (C) …
https://doi.org/10.7554/eLife.32032.023

Figure 5—figure supplement 2

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Changes in RpaB enrichment and downstream dusk gene expression after rapid changes in light intensity.

(A) Changes in enrichment of RpaB upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in target dusk gene expression (right heat map) for the 42 dusk …
https://doi.org/10.7554/eLife.32032.024

Figure 5—figure supplement 3

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Changes in RpaB and RNA polymerase enrichment upstream of dusk genes after rapid changes in light intensity.

(A) Changes in enrichment of RpaB upstream of dusk genes during High Light pulse conditions (left heat map) and corresponding changes in RNAP enrichment upstream of the same gene (right heat map) …
https://doi.org/10.7554/eLife.32032.025

Figure 6 with 1 supplement

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Global regulation of dusk gene expression in response to light changes.

(A) Number of dusk gene targets of RpaA only (red), RpaB only (blue), RpaA and RpaB (yellow), or neither (black). Target genes of binding sites of RpaA and RpaB were determined using chromatin …
https://doi.org/10.7554/eLife.32032.028

Figure 6—figure supplement 1

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Regulation of dusk sigma factor gene expression by RpaA and RpaB.

(A)-(C) Normalized ChIP-seq signal of RpaA (red), RpaB (blue), RNAP (green) and mock IP (black) upstream of the sigma factor genes (A) *rpoD6*, (B) *rpoD5*, and (C) *sigF2*. The location of the gene is …
https://doi.org/10.7554/eLife.32032.029

Figure 7 with 1 supplement

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Dusk genes group into three major clusters that show distinct and coordinated responses to changes in light intensity.

(A) Average expression profiles of genes belonging to the Early dusk gene cluster under Clear Day (magenta) and Shade pulse (gray) conditions (left y-axis). Dusk genes were grouped using k-means …
https://doi.org/10.7554/eLife.32032.030

Figure 7—source data 1 Lists of genes belonging to the Early, Middle, and Late dusk clusters, and scaled gene expression values.: https://doi.org/10.7554/eLife.32032.032
Download elife-32032-fig7-data1-v3.xlsx

Figure 7—figure supplement 1

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Average expression profiles of the major dusk gene clusters under various conditions.

(A) Average expression profiles of the Early (left plot), Middle (middle plot), and Late (right plot) dusk gene clusters under Low Light (black) and High Light pulse (orange) conditions (left …
https://doi.org/10.7554/eLife.32032.031

Figure 8 with 4 supplements

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Phenomenological modeling of the activation of clusters of light-responsive dusk genes.

(A) Normalized RpaA $\sim$ P levels (left plot) and RpaB $\sim$ P levels (right plot) under Clear Day (magenta) and Shade pulse (gray) conditions used as input for mathematical models of dusk gene expression. …
https://doi.org/10.7554/eLife.32032.033

Figure 8—figure supplement 1

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Best fit simulations of ‘RpaA-only’ and ‘RpaB-only’ models in which RpaA $\sim$ P or RpaB $\sim$ P solely activates the expression of the dusk gene clusters.

(A) Normalized RpaA $\sim$ P levels under Clear Day (magenta) and Shade pulse (gray) conditions used as input for mathematical models of dusk gene expression. RpaA $\sim$ P levels from all four light conditions …
https://doi.org/10.7554/eLife.32032.034

Figure 8—figure supplement 2

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Models in which either the Middle or Late cluster feeds back to influence Early cluster expression.

(A) Feedback model in which the expression of the Early dusk cluster is an activation Hill function of Middle gene expression and an activation Hill function of both RpaA $\sim$ P and RpaB $\sim$ P. The left …
https://doi.org/10.7554/eLife.32032.035

Figure 8—figure supplement 3

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Models in which either the Early or Late cluster feeds back to influence Middle cluster expression.

(A) Feedback model in which the expression of the Middle dusk cluster is an activation Hill function of Early gene expression and an activation Hill function of both RpaA $\sim$ P and RpaB $\sim$ P. The left …
https://doi.org/10.7554/eLife.32032.036

Figure 8—figure supplement 4

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Models in which either the Early or Middle cluster feeds back to influence Late cluster expression.

(A) Feedback model in which the expression of the Late dusk cluster is an activation Hill function of Early gene expression and an activation Hill function of both RpaA $\sim$ P and RpaB $\sim$ P. The left plot …
https://doi.org/10.7554/eLife.32032.037

Tables

Key resources table

Reagent type or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Synechococcus elongatus)	PCC 7942 (wild-type)	ATCC	Cat. Num. 33912
Strain, strain background (Escherichia coli)	Tuner (DE3)	EMD Millipore	Cat. Num. 70263
Gene (S. elongatus)	RNA polymerase Beta’ subunit	N/A	Cyanobase: Synpcc7942_1524
Gene (S. elongatus)	rpaB	N/A	Cyanobase: Synpcc7942_1453
Recombinant DNA reagent	RNA polymerase beta prime subunit FLAG	This paper	Addgene: 102337	Plasmid encoding C-terminal FLAG tag RNA polymerase Beta’ subunit (Synpcc7942_1524) with Kan selection marker, targeted to integrate at native gene locus
Recombinant DNA reagent	pET-48b(+)	EMD Millipore	Cat. Num. 71462
Renetic reagent (S. elongatus)	EOC 398 and EOC 399	This paper		S. elongatus PCC7942 transformed with RNA polymerase beta prime subunit FLAG plasmid. Confirmed by PCR and Western blot.
Antibody	anti-RpaB	This paper		Anti-RpaB serum was produced by Cocalico Biologicals. Anti-RpaB was affinity purified as described in this work.
Antibody	anti-RpaA	This paper		Anti-RpaA serum was produced by Cocalico Biologicals as described in Markson et al., 2013. Anti-RpaA was affinity purified as described in this work.
Antibody	FLAG M2 mouse monoclonal antibody	Sigma Aldrich	Cat. Num. F3165
Software, algorithm	Imagequant	GE Healthcare
Software, algorithm	Bowtie	PMID: 19261174
Software, algorithm	Peak-Seq	PMID: 19122651
Software, algorithm	MATLAB	MathWorks
Commercial assay or kit	RNeasy Mini kit	Qiagen	Cat. Num. 74104
Commercial assay or kit	Ribo-Zero bacteria rRNA removal kit	Illumina	Cat. Num. MRZMB126
Commercial assay or kit	Truseq Stranded mRNA sample prep kit	Illumina	Cat. Num. 20020594
Commercial assay or kit	NEBNext Ultra II DNA library prep kit	New England Biolabs	Cat. Num. E7645S
Chemical compound, drug	Phos-tagAcrylamide AAL-107	Wako Pure Chemical Industries	Cat. Num. 304–93521

Table 1

Fitting bounds.

Bounds used for fitting the variables in our simple model of gene expression. H is the Hill coefficient, $β$ is the max transcription rate, $α$ is the decay/dilution rate, $B$ is the background …

https://doi.org/10.7554/eLife.32032.038

Variable	Lower bound	Upper bound
H	0	7
$β$	0	80
$α$	0	80
$B$	0	10
$K$	0	1

Table 2

Fitting results.

The definitions of the variables are given in Equations 1-3, p. 1–3. The error is defined as the square root of the sum of the squared deviations between simulation and data.

https://doi.org/10.7554/eLife.32032.039

Model	Cluster	Figure	$B_{X}$	$β_{X}$	$α_{X}$	$K_{AX}$	$H_{AX}$	$K_{BX}$	$H_{BX}$	$K_{YX}$	$H_{YX}$	Error
RpaA-only	Early	7D	0.71	37.54	72.71	0.71	6.76	-	-	-	-	0.85
RpaB-only	Early	7-Fig. Supp. 2C	0.37	24.03	78.62	-	-	0.37	0.78	-	-	1.01
RpaA and RpaB	Early	7G	0.35	51.28	37.76	0.35	4.19	0.8	2.5	-	-	0.41
Feedback, M act.	Early	7-Fig. Supp. 3A	0.01	55.85	30.01	0.01	0.3	0.87	2.38	0.06	2.47	0.37
Feedback, M rep.	Early	7-Fig. Supp. 3B	0.67	58.69	38.89	0.67	6.96	0.62	2.47	0.96	7	0.24
Feedback, L act.	Early	7-Fig. Supp. 3C	0.2	35.87	19.03	0.2	4.43	0.98	3.35	0.05	6.15	0.38
Feedback, L rep.	Early	7I, 7-Fig. Supp. 3D	0.75	69.34	42.68	0.75	6.22	0.59	3.53	0.71	2.39	0.21
RpaA-only	Middle	7D	0.79	37.95	63	0.79	6.76	-	-	-	-	0.86
RpaB-only	Middle	7-Fig. Supp. 2C	0.26	0.03	-	-	-	0.26	5.6	-	-	0.85
RpaA and RpaB	Middle	7G	1	57.46	25.97	1	4.96	0.52	4.12	-	-	0.29
Feedback, E act.	Middle	7-Fig. Supp. 4A	0.8	23.73	22.19	0.8	6.96	0.49	4.53	0.21	6.35	0.32
Feedback, E rep.	Middle	7-Fig. Supp. 4B	0.73	71.08	39.24	0.73	5.14	0.53	6.58	0.74	0.88	0.35
Feedback, L act.	Middle	7I, 7-Fig. Supp. 4C	0.18	78.63	76.5	0.18	6.09	0.33	2.64	0.16	1.55	0.16
Feedback, L rep.	Middle	7-Fig. Supp. 4D	0.68	31.02	17.98	0.68	3.34	0.57	6.79	1	0	0.44
RpaA-only	Late	7D	0.96	39.82	64.37	0.96	6.7	-	-	-	-	0.78
RpaB-only	Late	7-Fig. Supp. 2C	0.05	0	0	-	-	0.05	0.68	-	-	0.79
RpaA and RpaB	Late	7G	0.95	77.65	67.1	0.95	7	0.48	5.9	-	-	0.5
Feedback, E act.	Late	7-Fig. Supp. 5A	0.99	23.93	20.01	0.99	5.8	0.4	6.95	0.18	6.77	0.53
Feedback, E rep.	Late	7-Fig. Supp. 5B	0.76	59.81	18.43	0.76	6.22	0.69	6.13	0.47	3.12	0.29
Feedback, M act.	Late	7I, 7-Fig. Supp. 5C	0.37	27.3	16.09	0.37	3.72	0.01	3.46	0.91	6.23	0.22
Feedback, M rep.	Late	7-Fig. Supp. 5D	0.86	25.1	14.46	0.86	6.92	0.48	7	1	0	0.52

Table 3

Parts for controllable light source.

The table includes the parts chosen for their specific properties. The remaining parts, such as wires, heat shrink tubing, thermal paste for mounting the LEDs on the heat sinks, proto-boards, and …

https://doi.org/10.7554/eLife.32032.040

Part name	Digikey part number	Current price ($)	Quantity
PWR SUP MEDICAL 18V 8.3A 150W	EPS439-ND	73.71	1
CONN RCPT 8CONT DIN SLD PNL MNT	SC2007-ND	5.64	1
LEDDynamics Flexblock BUCK BOOST 48V, 700 mA	788–1038-ND	19.99	4
AD7376 digital potentiometer	AD7376ARWZ10-ND	8.66	4
AC to DC power supply, 10VDC, 275 mA	993–1233-ND	4.68	2
BXRA-30E1200-B-03, Bridgelux, Warm white, LED	Not sold at Digikey.
	Need to order from:	10.47	4
	AMBIT ELECTRONICS, INC.
Aavid thermalloy Spotlight 47W heat sink	1061–1092-ND	9.50	4
Arduino Uno Board Rev3	1050–1024-ND	21.49	1

Table 4

Wiring the FlexBlock LED driver.

The FlexBlock LED driver needs to be connected in a ’boost only’ configuration (see spec sheet for more details), with connections as shown.

https://doi.org/10.7554/eLife.32032.041

Line	Connection
DIM GND	GND of 10 V power supply/Arduino
DIM	Wipe of AD7376 potentiometer (Pin 16)
Vin+	+of 18V power supply AND + of LED array
Vin-	GND of 18V power supply
LED+	NC (not connected)
LED-	- of LED array

Table 5

Wiring the AD7376 potentiometer.

We used the SOIC-16 housing for the AD7376 potentiometer for ease of soldering to wires. The table indicates how each pin was connected. The length of the GND wire from the Arduino board to the …

https://doi.org/10.7554/eLife.32032.042

Pin	Connection
1	+of 10 V power supply
2	GND (shared GND between that of 10V power supply and Arduino
3	GND
4	GND
5	pin 10 on Arduino (or any other pin designated as a Slave Select, such as 5, 6, or 9
6	+5V of Arduino
7	pin 13 on Arduino (SCLK)
8	NC (not connected)
9	NC
10	NC
11	pin 11 on Arduino (MOSI)
12	+5V of Arduino
13	NC
14	+of 10V power supply
15	NC
16	DIM line of FlexBlock

Additional files

Transparent reporting form: https://doi.org/10.7554/eLife.32032.043
Download elife-32032-transrepform-v3.pdf

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Cite this article

The circadian and light response pathways in cyanobacteria.

Natural clear day conditions sharpen the expression of dusk genes to peak just before expected darkness.

Figure 2—source data 1

Pigment levels of cyanobacteria grown under Low Light or Clear Day conditions reveal adjustments in the photosynthetic apparatus to optimize growth in different light conditions.

Gene expression dynamics of dusk and dawn circadian genes under Constant Light conditions (data from Markson et al., 2013).

Dawn gene expression increases during the early part of Clear Day relative to Low Light conditions.

The gene expression dynamics of glycogen production and breakdown enzymes change in Clear Day conditions relative to Low Light conditions.

Rapid changes in light intensity modulate the recruitment of RNA polymerase to dusk genes to control dusk gene expression.

Figure 3—source data 1

Figure 3—source data 2

Rapid changes in light intensity affect dawn gene expression in an opposite direction compared to dusk gene expression.

Changes in RNAP enrichment and downstream dusk gene expression after rapid changes in light intensity.

Changes in environmental light intensity regulate RpaA∼<math id="inf52"><mo mathvariant="normal">∼</mo></math>P DNA binding activity and RNAP recruitment to control dusk gene expression downstream of clock regulation of RpaA.

Figure 4—source data 1

Figure 4—source data 2

Representative Western blots used to quantify relative levels of RpaA∼<math id="inf57"><mo mathvariant="normal">∼</mo></math>P under dynamic light conditions.

Changes in RpaA enrichment and downstream dusk gene expression after rapid changes in light intensity.

Changes in RpaA and RNA polymerase enrichment upstream of dusk genes after rapid changes in light intensity.

Multifactorial behavior of RpaA∼<math id="inf68"><mo mathvariant="normal">∼</mo></math>P at select promoters under changes in light intensity.

Light-induced changes in RpaB∼<math id="inf84"><mo mathvariant="normal">∼</mo></math>P levels modulate RpaB and RNAP binding upstream of dusk genes to directly regulate dusk gene expression in response to light.

Figure 5—source data 1

Figure 5—source data 2

Representative Western blots used to quantify relative levels of RpaB∼<math id="inf89"><mo mathvariant="normal">∼</mo></math>P under dynamic light conditions.

Changes in RpaB enrichment and downstream dusk gene expression after rapid changes in light intensity.

Changes in RpaB and RNA polymerase enrichment upstream of dusk genes after rapid changes in light intensity.

Global regulation of dusk gene expression in response to light changes.

Regulation of dusk sigma factor gene expression by RpaA and RpaB.

Dusk genes group into three major clusters that show distinct and coordinated responses to changes in light intensity.

Figure 7—source data 1

Average expression profiles of the major dusk gene clusters under various conditions.

Phenomenological modeling of the activation of clusters of light-responsive dusk genes.

Best fit simulations of ‘RpaA-only’ and ‘RpaB-only’ models in which RpaA∼<math id="inf137"><mo mathvariant="normal">∼</mo></math>P or RpaB∼<math id="inf138"><mo mathvariant="normal">∼</mo></math>P solely activates the expression of the dusk gene clusters.

Models in which either the Middle or Late cluster feeds back to influence Early cluster expression.

Models in which either the Early or Late cluster feeds back to influence Middle cluster expression.

Models in which either the Early or Middle cluster feeds back to influence Late cluster expression.

Fitting bounds.

Fitting results.

Parts for controllable light source.

Wiring the FlexBlock LED driver.

Wiring the AD7376 potentiometer.

Transparent reporting form

Changes in environmental light intensity regulate RpaA $\sim$ P DNA binding activity and RNAP recruitment to control dusk gene expression downstream of clock regulation of RpaA.

Representative Western blots used to quantify relative levels of RpaA $\sim$ P under dynamic light conditions.

Multifactorial behavior of RpaA $\sim$ P at select promoters under changes in light intensity.

Light-induced changes in RpaB $\sim$ P levels modulate RpaB and RNAP binding upstream of dusk genes to directly regulate dusk gene expression in response to light.

Representative Western blots used to quantify relative levels of RpaB $\sim$ P under dynamic light conditions.

Best fit simulations of ‘RpaA-only’ and ‘RpaB-only’ models in which RpaA $\sim$ P or RpaB $\sim$ P solely activates the expression of the dusk gene clusters.