1
2
3
4
An evolutionarily conserved RNase-based mechanism for repression of transcriptional
positive autoregulation
5
Nitin S. Baliga
Elisabeth J. Wurtmann, Alexander V. Ratushny, Min Pan, Karlyn D. Beer, John D. Aitchison,
6
7
Supplementary Material Contents:
8
-
Figures S1-S8
9
-
Tables S1-S4
10
-
Supplementary Experimental Procedures
11
-
Supplementary References
12
13
Supplementary File 1. Gene expression in strains lacking RNase E, G, R, II, III, or
14
polynucleotide phosphorylase (PNPase) (Lee et al., 2002; Mohanty and Kushner, 2003; Stead et
15
al., 2011; Phadtare, 2012). See .xlsx spreadsheet file for details.
16
2
17
18
Figure S1. Conservation of active site residues between the mammalian L-PSP ribonuclease and
19
H. salinarum VNG2099C.
20
The H. salinarum VNG2099C protein sequence was aligned to mammalian and Sulfolobus
21
tokodaii YjgF/L-PSP protein sequences. Secondary and tertiary structures identified in the
22
crystal structure of the S. tokodaii protein are displayed for -strands (arrows), - and 310-helices
23
(helices), inter-subunit clefts proposed to be catalytic sites (), and the conserved cavity (•)
24
(Miyakawa et al., 2006). Conserved residues are boxed, with identical residues in white letters
25
and similar residues in red letters. The alignment was determined using SwissProt and displayed
26
with ESPript (Gouet et al., 2003).
27
3
28
29
Figure S2. Growth analysis of H. salinarum strains lacking ribonuclease orthologs.
30
Growth of strains lacking the (A) RNase Zs ortholog VNG1503C, (B) RNase CPSF73 ortholog
31
VNG2512G, or (C) RNase R ortholog VNG2647G compared to the parent ura3 strain. Error
32
bars, s.e.m. n = 4-6 biological replicates per strain.
33
4
34
35
Figure S3. Sampling for ura3 and 2099 genome-wide expression analysis.
36
Sampling for total RNA extraction and gene expression analysis (Figure 1C) occurred at four
37
points of batch culture growth from the ura3 and 2099 strains (mid-log, late-log, early
38
stationary, and late stationary; arrows). The log phase samples were taken at the equivalent
39
OD600 values for the ura3 and 2099 strains. As the carrying capacity differs for the two
40
strains, stationary phase points were determined as occurring after an equivalent proportion of
41
the duration of stationary phase.
42
5
43
44
Figure S4. VNG2099C associates with the kdpQ mRNA in vivo.
45
(A) Lysates from mid-log phase cultures of the Pfdx_VNG2099C_CHA and Pfdx_tbpB_CHA (C-
46
terminal HA-tagged) strains were subjected to immunoprecipitation with anti-HA antibodies.
47
Immunoprecipitates were separated by SDS-PAGE and transferred to a membrane for
48
immunoblotting using anti-HA antibodies to verify expression and immunoprecipitation of the
49
tagged proteins, VNG2099C-CHA and TbpB-CHA. (B) Quantitative reverse transcriptase RT-
50
PCR of RNA immunoprecipitated from formaldehyde-crosslinked cells using anti-HA
51
antibodies. The relative co-immunoprecipitation efficiency provides the level of the kdpQ
52
mRNA qRT-PCR product after immunoprecipitation relative to before immunoprecipitation,
53
with the ratio in the control strain (transformed with the plasmid pMTF_Pfdx_CHA) defined as 1.
54
Fold differences in RNA levels were calculated by the Ct method using the glucose kinase
55
gene (glcK; VNG2629G) as a reference gene. Immunoprecipitation was also performed in a
56
strain expressing an HA-tagged transcription factor, TbpB, as a control; co-immunoprecipitation
57
of kdpQ mRNA from that strain was not significantly different from the pMTF_Pfdx_CHA strain.
6
58
Error bars, s.e.m.; the significant P-value as determined by a t-test is listed, n = 4 biological
59
replicates per strain.
60
7
61
62
Figure S5. Expression levels of VNG2099C are anti-correlated with its target genes.
63
Correlation between VNG2099C and all other genes was calculated across the previous dataset of
64
relative expression changes in 1,495 environmental conditions (Reiss D.J., Brooks A.N., &
65
Baliga N.S., unpublished; Bonneau et al., 2007). The 23 genes identified as VNG2099C targets
66
by expression analysis in the 2099 strain (Figure 3B) are statistically enriched among the genes
67
with correlation < -0.55 as calculated by hypergeometric testing.
68
8
69
70
Figure S6. Expression levels of VNG2099C are anti-correlated to corresponding levels of kdp,
71
bop, and yhdG.
72
(A) VNG2099C expression decreases from log to stationary phase while expression of kdp, the
73
bacteriorhodopsin gene bop, and the ornithine-arginine transport gene yhdG increase. For log
74
phase (OD600 < 1.0; n = 44) and stationary phase points (OD600 > 1.0; n = 20) of growth curve
75
experiments (Reiss D.J., Brooks A.N., & Baliga N.S., unpublished; Bonneau et al., 2007),
76
expression values are given as log10 ratios relative to a mid-log reference sample. Expression
77
values of kdp genes are the median of the values for kdpA, kdpB, kdpC, and kdpQ. Significance
78
of expression change differences between conditions was determined using two-sample t-tests,
79
and P-values are listed. (B-D) The relative changes in mRNA expression level of the kdp, bop,
80
and yhdG genes are plotted against relative changes in expression level of VNG2099C from a
9
81
previous dataset of 1,495 environmental conditions (Reiss D.J., Brooks A.N., & Baliga N.S.,
82
unpublished; Bonneau et al., 2007). Points in log phase are colored red; points in stationary
83
phase are colored green. The boxes indicate the subset of points where both VNG2099C and
84
target gene expression > 1.5 standard deviations from the median. (B) Correlation coefficient: -
85
0.63, R2 = 0.40, P < 2.2 x 10-16. Within the low VNG2099C, high kdp subset, the environmental
86
ontology terms light and low oxygen are significantly enriched (P = 1 x 10-3, P = 1 x 10-3).
87
Within the high VNG2099C, low kdp subset, the term darkness is significantly enriched (P =
88
0.01). (C) Correlation coefficient: -0.32, R2 = 0.10, P < 2.2 x 10-16. Within the low VNG2099C,
89
high bop subset, the term light is significantly enriched (P-value: 4 x 10-4). (D) Correlation
90
coefficient: -0.60, R2 = 0.36, P < 2.2 x 10-16. Within the low VNG2099C, high yhdG subset, the
91
terms light and low oxygen are significantly enriched (P = 1 x 10-3, P = 1 x 10-3). Within the
92
high VNG2099C, low yhdG subset, the term darkness is significantly enriched (P = 0.01).
93
10
94
95
Figure S7. Co-regulation of VNG2099C and aerobic metabolism genes.
96
VNG2099C is found in a conditionally co-regulated module (‘corem’) with genes involved in the
97
TCA cycle, pyruvate metabolism, and the electron transport chain. Genes that are putatively co-
98
regulated over subsets of experimental conditions were detected by an ensemble implementation
11
99
of the cMonkey algorithm, which incorporates clustering of gene expression data, gene
100
functional associations, and de novo sequence motif detection (Reiss D.J., Brooks A.N., &
101
Baliga N.S., unpublished; Bonneau et al., 2007). (A) The median change in mRNA expression
102
level of the corem genes relative to the mid-log reference condition are plotted from a previous
103
dataset of 1,495 environmental conditions (Reiss D.J., Brooks A.N., & Baliga N.S., unpublished;
104
Bonneau et al., 2007). The corem genes were calculated to be co-regulated over the first 447
105
conditions. Error bars, s.d. (B) The corem member genes. (C) Enriched functional annotation
106
clusters for the corem member genes are listed with Benjamini-Hochberg corrected P-values. (D)
107
The motif logo for a gene regulatory element that is associated with the corem and VNG2099C (q
108
= 3 x 10-3, hypergeometric test with Benjamini-Hochberg correction).
109
12
110
111
Figure S8. Effect of the kdp operon on cellular ATP level.
112
Intracellular ATP levels are elevated in the kdp strain relative to the ura3 parental strain (P =
113
7 x 10-2, t-test, n = 5 biological replicates; error bars, s.e.m.). The intracellular ATP level of each
114
strain was normalized to the level of the ura3 parental strain.
115
13
116
RNase type
NRC-1 gene NRC-1 GI
notes
BHB
NRC-1 ortholog
locus
VNG2210G
endA
15791035
CPSF73
CPSF73
Dom34/Pelota
IF5a
VNG0401G
VNG2512G
VNG1506G
VNG1768G
epf2
epf1
pelA
eif5a
15789650
15791270
15790498
15790688
tRNA splicing
endonuclease
aCPSF1
aCPSF2
L-PSP
RNase E/G
RNase H type I
RNase H type II
RNase J
RNase P
RNase P
VNG2099C
VNG2627C
VNG0255C
VNG1984G
VNG1149Cm
VNGs01
VNG1279H
RNase P
VNG0599C
15789807
RNase P
VNG1699C
15790640
RNase P
VNG1281H
15790328
RNase P
VNG1157G
rpl7ae
15790233
RNase R/II
RNase Zl
RNase Zs
VNG2647G
VNG2239C
VNG1503C
vacB
15791376
15791057
15790496
rnhB
homology to eukaryotic
initiation factor 5A; RNase
activity demonstrated by
Wagner and Klug (2007)
15790939
15791358
15789547
15790853
16554486
rnpB
15790326
RNase P RNA
homology to eukaryotic
core protein hPop5/Pop5
homology to eukaryotic
core protein Rpp21/Rpr2
homology to eukaryotic
core protein Rpp29/Pop4
homology to eukaryotic
core protein Rpp30/Rpp1
homology to eukaryotic
core protein Rpp38/L7Ae
117
118
Table S1. Putative H. salinarum ribonuclease orthologs.
119
Putative ribonuclease orthologs in H. salinarum NRC-1 were identified from genome
120
annotations as well as by manual curation using BLAST searching for orthologs of ribonucleases
121
described in other species.
122
14
123
Media
2:92
26:74
100:0
[Na+]
4.326
4.326
4.326
[Cl-]
4.294
4.321
4.402
[Mg2+]
0.108
0.081
0.000
[SO2-]
0.109
0.082
0.001
[K+]
0.002
0.029
0.110
[total] (M)
8.839
8.839
8.839
124
125
Table S2. Media composition.
126
Media are named by the [KCl]:[MgSO4] ratio. Ion molarity was calculated from the added salts
127
and the measured contribution of ions from the citrate and peptone reagents. Ion analysis for Na,
128
Cl, Mg, SO4, and K ion levels in the the invariant rich media base (3 g l-1 sodium citrate + 10 g l-
129
1
130
methods were used to measure the concentrations of each ion: sodium and potassium – atomic
131
absorption spectroscopy; chloride – titration with 0.01 N silver nitrate; magnesium – titration
132
with 0.01 M EDTA; sulfate – gravimetric precipitation with barium chloride. Selected samples
133
were submitted in blind triplicate to obtain a measure of technical variability. Coefficients of
134
variation ranged from 0 to 5.5%.
135
Oxoid peptone) was performed by Hoh Pak Laboratories, Inc (New Iberia, LA). The following
15
136
Gene
kdpA
kdpB
kdpC
trkA2
VNG1238C
VNG6180H
boa4
bop
yhdG
cat4
VNG6183H
VNG6185H
rhl
VNG0335B
VNG6039H
npa
VNG0013C
VNG0026C
VNG0217H
VNG1734H
VNG6165H
VNG6347H
VNG6365H
Function
K+-transporting ATPase A chain
K+-transporting ATPase B chain
K+-transporting ATPase C chain
Trk K+ uptake system, NADH binding protein
TrkA-domain protein
Putative anion exchange protein
Bacterio-opsin-like protein
Bacteriorhodopsin precursor
Putative ornithine-arginine transporter
Cationic amino acid transporter
Cationic amino acid transporter
Cationic amino acid transporter
Putative DNA helicase
Purine nucleotide phosphorylase
Putative restriction enzyme
Putative transposase
Putative transposase
Putative transposase
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
System
Ion transport: K+ high-affinity
Ion transport: K+ high-affinity
Ion transport: K+ high-affinity
Ion transport: K+ low-affinity
Ion transport: K+ low-affinity
Ion transport: other
Phototrophy
Phototrophy
Amino acid transport
Amino acid transport
Amino acid transport
Amino acid transport
DNA processes
DNA processes
DNA processes
DNA processes
DNA processes
DNA processes
Unknown
Unknown
Unknown
Unknown
Unknown
137
138
Table S3. Genes dysregulated in the 2099 strain.
139
Genes with significantly increased expression in the 2099 strain as identified by significance
140
analysis for microarrays (SAM) (Saeed et al., 2006) using an FDR cut-off of 10%.
141
16
142
Function
Carbon transport/metabolism
Membrane signal
transduction
Transcriptional regulation
Transport
Positively
autoregulated
TF
fhlA
fucR
gutM
tdcA
xylR
evgA
RNase-targeted
transcript
RNase(s)
ATP
hydrolysis
GTP
hydrolysis
fhlA-hypABCDE
fucIKPRU
gutMQ-srlABDER
tdcABCDEFG
xylFGHR
evgAS
PNPase
PNPase, RNase II
PNPase
PNPase
PNPase, RNase II
PNPase
FhlA, HypE
FucK
GutQ
HypB
yeiL
baeR
marA
yeiL
baeRS-mdtABCD
marABR
PNPase
PNPase
RNase II
XylG
EvgS
BaeS, MdtA
143
144
Table S4. RNase regulation of positively-autoregulated operons in E. coli.
145
E. coli operon membership was retrieved from RegulonDB (Salgado et al., 2012). Putative
146
RNase targets were determined as the union of genes with ≥ 2.5-fold increased expression in
147
strains lacking RNase E, G, R, II, III, or polynucleotide phosphorylase (PNPase) (Lee et al.,
148
2002; Mohanty and Kushner, 2003; Stead et al., 2011; Phadtare, 2012; Supplementary File 1).
149
The prominence of PNPase as a regulator of these operons may reflect the general prominence of
150
PNPase as a regulator of mRNA decay. Specifically, there are 503 putative targets for PNPase,
151
229 for RNase II, 66 for RNase E, 19 for RNase R, 9 for RNase III, and 2 for RNase G.
152
Within the positively-autoregulated operons regulated by RNases, many genes have
153
putative or demonstrated ATPase or GTPase activity: FhlA (Hopper and Böck, 1995), HypB
154
(Maier et al., 1995), HypE (Blokesch et al., 2004), FucK (Bork et al., 1992), GutQ (Ramseier et
155
al., 1994), XylG (Song and Park, 1998), EvgS (Perraud et al., 1998), BaeS (Nagasawa et al.,
156
1993), and MdtA (Perreten et al., 2001).
157
158
17
159
Supplementary Experimental Procedures
160
Strains
161
The Pfdx-2099-CHA plasmid was used to express VNG2099C with a C-terminal HA epitope from
162
the ferredoxin gene promoter in the NRC-1 strain. This plasmid was constructed by insertion of
163
the
164
GGCCGGCATATGAAGCGCCCGATTGAAACC-3’)
165
GGCCGGAAGCTTGTCGCGCTGTGCGGCGATGG-3’) primers into the pMTF_Pfdx_CHA
166
plasmid digested with NdeI and HindIII. Similarly, the Pfdx-tbpB-CHA plasmid was used to
167
express tbpB with a C-terminal HA epitope from the ferredoxin gene promoter in the NRC-1
168
strain. The plasmid was created by amplifying the tbpB gene sequence with the forward (5’-
169
GCACCATATGAGCACGCTGGCGGACACGATCCATA-3’)
170
GCACAAGCTTTCAGTCAAGCAGGCCGAGTTCGGTGAGTCGG-3’) primers for insertion
171
into the pMTF-Pfdx_CHA plasmid digested with NdeI and HindIII. The Pfdx-2099-CHA, Pfdx-
172
tbpB-CHA, or pMTF_Pfdx_CHA plasmid (control) were transformed into the NRC-1 strain.
173
Following transformation, the strains were cultured with 0.02 mg ml-1 Mevinolin to select for
174
plasmid maintenance.
175
176
VNG2099C
gene
amplified
by
and
forward
reverse
and
reverse
(5’-
(5’-
(5’-
The kdpFABCQ strain was created from the ura3 strain using unmarked, in-frame
deletion as described in the Experimental Procedures.
177
178
sequence
Crosslinked immunoprecipitation
18
179
Cells grown to mid-log phase were crosslinked using 1% formaldehyde for 15’ at 37°C followed
180
by quenching with 0.125M glycine for 5’ at 37°C. Cells (12.5 OD600 units) were then pelleted
181
and lysed by sonication in 0.5 ml lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM
182
EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxychlolate) plus 40 U RNase inhibitor
183
(Ambion). Lysates were cleared by centrifugation at 10k x g for 10’ at 4°C prior to incubation
184
with anti-HA antibodies (4.6 ul of 1.6 ug/ul slurry, Thermo Scientific) overnight at 4°C with
185
rotation. Immunoprecipitates were washed three times in lysis buffer. RNA-protein crosslinks
186
were reversed in immunoprecipitate and input samples by incubating at 70°C for 45’ in elution
187
buffer (50 mM Tris-HCl pH 7.4, 5 mM EDTA, 10 mM DTT, 1% SDS) followed by
188
phenol:chloroform extraction and ethanol precipitation. RNA pellets were resuspended in H2O,
189
treated with RNase-free DNase (Promega), and RNA quality and quantity was determined using
190
a Nanodrop spectrophotometer (Thermo Fisher Scientific). Reverse transcription qRT–PCR
191
analyses were performed as described in Experimental Procedures. Four biological replicates
192
were used, with each qRT-PCR reaction performed in 2-3 technical replicates.
193
194
19
195
Supplementary References
196
197
198
Blokesch, M., Paschos, A., Bauer, A., Reissmann, S., Drapal, N., and Böck, A. (2004) Analysis
of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation
proteins HypF and HypE. Eur J Biochem 271: 3428–3436.
199
200
201
Bonneau, R., Facciotti, M.T., Reiss, D.J., Schmid, A.K., Pan, M., Kaur, A., et al. (2007) A
predictive model for transcriptional control of physiology in a free living cell. Cell 131: 1354–
1365.
202
203
204
Bork, P., Sander, C., and Valencia, A. (1992) An ATPase domain common to prokaryotic cell
cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci USA 89:
7290–7294.
205
206
207
Gouet, P., Robert, X., and Courcelle, E. (2003) ESPript/ENDscript: extracting and rendering
sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31: 3320–
3323.
208
209
Hopper, S., and Böck, A. (1995) Effector-mediated stimulation of ATPase activity by the sigma
54-dependent transcriptional activator FHLA from Escherichia coli. J Bacteriol 177: 2798–2803.
210
211
Maier, T., Lottspeich, F., and Böck, A. (1995) GTP hydrolysis by HypB is essential for nickel
insertion into hydrogenases of Escherichia coli. Eur J Biochem 230: 133–138.
212
213
214
Miyakawa, T., Lee, W.C., Hatano, K., Kato, Y., Sawano, Y., Miyazono, K., et al. (2006) Crystal
structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic archaeon
Sulfolobus tokodaii strain 7. Proteins 62: 557–561.
215
216
Nagasawa, S., Ishige, K., and Mizuno, T. (1993) Novel members of the two-component signal
transduction genes in Escherichia coli. J Biochem 114: 350–357.
217
218
219
Perraud, A.L., Kimmel, B., Weiss, V., and Gross, R. (1998) Specificity of the BvgAS and
EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins. Mol
Microbiol 27: 875–887.
220
221
222
Perreten, V., Schwarz, F.V., Teuber, M., and Levy, S.B. (2001) Mdt(A), a New Efflux Protein
Conferring Multiple Antibiotic Resistance in Lactococcus lactis and Escherichia coli. Antimicrob
Agents Chemother 45: 1109–1114.
223
224
225
Ramseier, T.M., Figge, R.M., and Saier, M.H., Jr (1994) DNA sequence of a gene in Escherichia
coli encoding a putative tripartite transcription factor with receiver, ATPase and DNA binding
domains. DNA Seq 5: 17–24.
226
227
Saeed, A.I., Bhagabati, N.K., Braisted, J.C., Liang, W., Sharov, V., Howe, E.A., et al. (2006)
TM4 Microarray Software Suite. Methods Enzymol 411: 134–193.
228
229
Song, S., and Park, C. (1998) Utilization of d-ribose through d-xylose transporter. FEMS
Microbiol Lett 163: 255–261.
20
230
231
Wagner, S., and Klug, G. (2007) An archaeal protein with homology to the eukaryotic translation
initiation factor 5A shows ribonucleolytic activity. J Biol Chem 282: 13966-13976.