Transcript mmi12384-sup-0001-si

Table S1 Strains, plasmids and oligonucleotides
Strain/vectors/Primers
Description
Reference
M. smegmatis mc2
Wild type strain
M.tuberculosis H37Ra
(A non virlulent laboratorystrain of M .tuberculosis)
E.coli DH10B
Δ(mrr-hsd RMS-mcrBC) mcrA recA1 Laboratory
stock
pARN104
A derivative of pUC18
Tare et.al.,2012
pJam2
Expression vector for mycobacteria
Triccas et al., 1998
PrrnPCL1 sense
5’TCACCTATGGATATCTATGGATGACCGAACC
TGGTCTTGACTCCATTGCCGGATTTGTATTAG
ACTGGCAGGGTCGCCCCGAAGCGGGCGG 3’
PrrnPCL1 antisense
5’CCGCCCGCTTCGGGGCGACCCTGCCAGTCTA
ATACAAATCCGGCAATGGAGTCAAGACCAGG
TTCGGTCATCCATAGATATCCATAGGTGA3’
PrrnPCL1 sense
5’TCACCTATGGATATCTATGGATGACCGAACC
TGGTCTTGACTCCATTGCCGGATTTGTATTAG
ACCGGCAGGGTCGCCCCGAAGCGGGCGG3’
PrrnPCL1 antisense
5’CCGCCCGCTTCGGGGCGACCCTGCCGGTCTA
ATACAAATCCGGCAATGGAGTCAAGACCAGG
TTCGGTCATCCATAGATATCCATAGGTGA3’
Wt SigA ntd Rev
5’GTTCGCGCCTACCTCAAACAGATCG3’
Wt SigA ctd For
5’CGATCTGTTTCGAGTAGGCGCGAAC3’
L232M ntd Rev
5’GTTCGCGCCTACATGAAACAGATCG3’
L232M ctd For
5’CGATCTGTTTCATGTAGGCGCGAAC3’
SigFL For
5’GCCTCTAGAGTAACGACCGAAGGGGTGTATG
TGGCAG3’
5’CAGAGTCTTCAAAGTCCAGGTAGTCGCGC3’
SigFL Rev
Tare et.al.,2012
This work
1
Table S2 Representative bacteria in two groups based on residues in rrn discriminator and the
sigma 1.2 region
Group A
rrn
(Sigma)
M. tuberculosis
T
L
M. smegmatis
T
L
B. subtilis
T
L
L. monocytogenes
T
L
S. aureus
T
L
S. pneumoniae
T
L
E. coli
C
M
S. enteritica
C
M
S. Typhimurium
C
M
V. cholerae
C
M
Group B
2
Supplementary Fig. S1
A
Promoters responsive to growth phase dependent regulation
Promoters
-35----------------spacer-----------
-10 –discriminator+1
3ds
M.tb PgyrB1
TCGGCCctggcgcccgatcacgg—-TACAGTggTgtgc-G
M.tb PrrnpcL1
TTGACTccattgccggat-tt-g--TATTAGacTggca-G
M.tb PrrnAP1
TCGGTGccgagattcgaacg--g--TATGCTgtTaggc-G
M.tb PrpsL
TTGACCtgccagactggcggcgggTATTGTggTtgctcG
M.sm PrrnBP1
TTGACTcccagtttccaaggacg--TAACTTatTccag-G
M.sm PrrnAP1
TCGGAGccgagagagagccga—g--TAAGCTcgT--ag-G
Promoters non-responsive to growth phase dependent regulation
B
M.tb PmetU
TTGGCGagcttcgtgcgtgttcgg—TAGCCTggCattt-A
M.smeg Pgyr
TCGGTGctgtcgctatctcgcgg—-TAGACTggAcgac-G
M.tb PgroEL2
TGCACTcggcatagagtgct----AAGAATaaCgttgG
PrrnPCL1
Supplementary Fig. S1(A) Sequence of promoters from genus mycobacteria responsive to growth phase
dependent control and having conserved thymine ( upper case and underlined) at three nucleotide
downstream (3ds) to -10 element. -35,-10 elements and start site are in upper case. The sequences of the
promoters not responsive to the growth phase dependent control are also shown. (B) The effect of mutation
of the base two nucleotides downstream to -10 element (2ntds) on the iNTPs mediated regulation of PrrnPCL1
3
. The amount of transcripts formed in presence of iNTPs are normalized to that obtained in absence of the
molecules. AU represents fold change on Y axis.
Supplementary Fig. S2
A
Km
190
22
30
194 µM
200
PrrnPCL1
KGTP
150
1- T3ntds: L232SigA-RNAP
2- C3ntds: L232SigA-RNAP
3- T3ntds: L232MSigA-RNAP
4- C3ntds:L232MSigA-RNAP
100
50
0
B
Km
1
2
3
4
206
31
43
181 µM
PgyrB1
KGTP,ATP
200
150
100
1- T3ntds : L232SigA-RNAP
2- C3ntds : L232SigA-RNAP
3- T3ntds : L232MSigA-RNAP
4- C3ntds : L232MSigA-RNAP
50
0
1
2
3
4
Supplementary Fig. S2 Determination of KiNTP . (A) KiNTP values of PrrnPCL1 and its mutant in discriminator. (KGTP)
(B) KiNTP values of PgyrB1 and its mutant in discriminator (K GTP, ATP ) are shown. T3ntds, C3ntds promoters were
transcribed with wild type (L232SigA) and mutant RNAP (L232M SigA) in separate reactions as described in the
panel right to the graph. The Km values (KGTP for PrrnPCl1, KGTP,ATP for PgyrB1) were plotted on the Y axis of the graphs.
The concentrations of the iNTPs used in the reactions were 20, 50, 100, 200, 400, 800 and 1000µM.
4
Supplementary Fig. S3.
A
PmetU
C
L232M RNAP
M.tb
-35
-10
dis +1
gcgatTTGGCGagcttcgtgcgtgttcggTAGCCTggcatttAccg
3ds
L232 RNAP
B
Supplementary Fig. S3. (A) Alignment of aminoacids in the stretch of 1.2 region of principle sigma factors from
different bacteria. Sequences are taken from NCBI (National Centre for Biotechnology Information) and aligned
using ClustalW. The residues homologous to M102 in 1.2 region of Sig70 of E. coli is highlighted. E. coliEscherichia coli, S. typhi-Salmonella typhi, P. aeruginosa-Pseudomonas aeruginosa, R. sphareoides -Rhodobacter
sphareoides, C. crescentus- Caulobacter crescentus, L. monocyto- Lactobacillus monocyotgenes, B. subtilli-Bacillus
subtillis; S. aureus- Staphylococcus aureus, S. pnemoni- Streptococcus pnuemoniae; N. brasilie –Nocardia
brasiliensis, S. coelicol- Streptococcus coelicolor, M. smegmatis- Mycobacterium smegmatis; M. lepraeMycobacterium leprae, M. tubercul- Mycobacterium tuberculosis, S. aureofac- Staphylococcus aureofaciens., L.
lactis –Lactobacillus lactis, C. acetobut- Clostridium acetobutylicum. (B) The sequence of PmetU -35, -10 elements
and start site is in upper case. Base at three nucleotide downstream (3ds) is marked bold and underlined. (C )In vitro
transcription at iNTP insensitive promoter, PmetU to check the efficiency of RNAPs containing L232 and L232M
SigA.
5
Supplementary Fig. S4
A
Non- template
Template
ntds
34
CG
GC
CT
GA
Fold Change
B
1.0
CG: L232 SigA-RNAP
CT: L232 SigA-RNAP
0.8
CT: L232M SigA-RNAP
CG: L232M SigA-RNAP
0.6
0.4
0.2
0.0
0
10
20
30
C
Promoter
SigA
koff
C3ntds
L232
0.5
C3ntds
L232M 3.5
C3T4ntds
L232
C3T4ntds
L232M 2.5
0.65
40
Time (min)
Supplementary Fig. S4 Contribution of nucleotide at 4ntds in stability of open complex. (A). Templates
used in the assay. The nucleotides at 3ntds and 4 ntds in different templates used are shown. Stability of
open complexes at PrrnPCl1 templates having mutation at 3ntds and/or 3 and 4 ntds (ntds- nucleotides
downstream to -10 element). (B). The stability of open complexes were determined by monitoring the time
dependent dissociation RNAP using filter binding assay. The radioactivity counts in the filters spotted with
reactions at different time-points were normalized to that obtained at zero time point. (C). koff was
determined by single phase exponential decay analysis using Graph pad ver. 2.3. The SigA (wild type
/mutant) present in the holo-RNAP in the reaction is shown in the table.
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Supplementary Fig. S5
A
T3ntds PrrnPCL1
C3ntds PrrnPCL1
3ntds
T
A
C
G
Non- template
Template
complementary base pair
at 3ntds
Bubble templates
CT PrrnPCL1
C
T
CC PrrnPCL1
C
C
CA PrrnPCL1
C
TT PrrnPCL1
C
T
A
mis-match at 3ntds
T
B
C
Promoter
Relative
koff
T3ntds
1.06
C3ntds
0.16
Bubble C
CT
0.15
CC
0.2
CA
0.45
Supplementary Fig.S5 Contribution of cytosine in non-template strand to open complex stability (A).
Representation of various templates used in the assay. Templates with a complementary base-pairing at
three nucleotide down stream (3ntds) to -10 are labeled as T3ntds (wild type PrrnPCL1) and C3ntds (mutant
PrrnPCL1). Templates having a mis-match at 3ntds position are labeled as bubble templates. (B). Stability of
open complexes at bubble templates. The stability of open complexes were determined by monitoring the
time dependent dissociation RNAP using filter binding assay. The radioactivity counts in the filters spotted
with reactions at different time-points were normalized to that obtained at zero time point. (C). koff was
determined by single phase exponential decay analysis using Graph pad ver.2.3. The values are normalized
to the koff of TT bubble and referred as relative koff. Bubble C- non-template strand with C in non-template
strand annealed with templates having A, T or C [refer (A)].
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Supplementary Fig. S6
A
T3ntds PrrnPCL1
C3ntds PrrnPCL1
3ntds
T
A
C
G
Non- template
Template
complementary base pair
at 3ntds
Bubble templates
T
TG PrrnPCL1
G
G
GG PrrnPCL1
AG PrrnPCL1
G
mis-match at 3ntds
A
G
TT PrrnPCL1
B
T
T
C
Promoter
Relative
koff
T3ntds
1.06
C3ntds
0.16
Bubble G
TG
1
AG
0.96
GG
0.86
Supplementary Fig. S6 Contribution of G in template strand in open complex stability. (A)
Representation of various templates used in the assay. Templates with a complementary base-pairing at
three nucleotide down stream (3ntds) to -10 are labeled as T3ntds (wild type PrrnPCL1) and C3ntds (mutant
PrrnPCL1). Templates having a mis-match at 3ntds position are labeled as bubble templates. Stability of
open complexes at G bubble templates. (B) The stability of the open complexes were determined as
described in legend to Supplementary Fig. S5. The radioactivity counts in the filters spotted with reactions
at different time-points were normalized to that obtained at zero time point. (C) koff was determined by
single phase exponential decay analysis using Graph pad ver.2.3. The values are normalized to the koff of
TT bubble. Bubble G- non-template strand with G in template strand annealed with non template strand
having A, T or C (refer A).
8
Supplementary Fig. S7.
E.coli
V.cho
S.ent
S.typhim
M.tub
M.sm
B.su
L.mon
S.aue
S.pn
-35
-10
2ds
+1
TTGTCA------- TATAATgcgccaccA
TTGACA------- TATAATccgccctcA
TTGTCT--------TATAATgcgcctccA
TGTCTT--------TATATTgcgcctccA
TTGACT--------TATTAGacTggca-G
TTGACT--------TAACTTatTccag-G
TTGCAA--------TATATTatTaaac-G
TTGCAA--------TATATTtaTaaac-G
TTGAAA--------TAAAATaaTattt-G
TTGACA--------TATAATagTaaga-G
3ds
Supplementary Fig.S7 Sequences of rrn promoters from representative bacteria of gram
positive and gram negative groups. -35 and -10 elements are in upper case and underlined.
Nucleotide at two nucleotide downstream to -10 element (2ds) is marked bold and underlined in
promoters of gram negative bacteria. Promoters from gram positive having conserved thymine
at (3ds) three nucleotides downstream to -10 element (upper case, bold and underlined) are also
listed. Sequences are taken from (32).
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