RsgA releases RbfA from 30S ribosomal subunit during a late
stage of ribosome biosynthesis
Simon Goto, Shingo Kato, Takatsugu Kimura, Akira Muto and Hyouta Himeno
A
metY
nusA
infB
rbfA truB rpsO
pnp
yhbM
yhbC
652bp
2427bp
B
P30L
D100G
G77D
G84E
R10H
Supplementary Figure 1: Distribution of rbfA-suppressing mutations.
(A) Genomic context around rsgA-suppressing mutations.
The metY operon is illustrated. The region marked as “2427 bp” was inserted i¡nto pUC26-2, a plasmid clone
selected for rsgA-suppressing activity. The region marked as “652 bp” was subcloned into a low-copy plasmid
and confirmed to retain rsgA-suppressing activity.
(B) Distribution of rsgA-suppressing mutations in the tertiary structure of E. coli RbfA.
Five out of eight mutations are indicated in an NMR structure, PDB code 1KKG (Huang et al, 2003). The NMR
structure renders truncated RbfA lacking C-terminal 25 residues, so that the other three mutations within the
C-terminal region are not indicated. This figure was generated using MolScript (Kraulis, 1991).
1
A
OD600
B
1
pMWrsgA
pMWrbfA1
pMWrbfA4
pMWrbfA11
pMWrbfA15
pMWrbfA26
pMWrbfA107
pMWrbfA121
pMWrbfA124
pMWrbfA
pMW118
+
0
1
2
3
4
5
6
4
5
6
Time (hour)
C
OD600
+
pMWrbfA
pMWrbfA1
pMWrbfA4
pMWrbfA11
pMWrbfA15
pMWrbfA26
pMWrbfA107
pMWrbfA121
pMWrbfA124
1
pMW118
0
1
2
3
Time (hour)
Supplementary Figure 2: Mutations in rbfA restore cell growth.
Growth rates of (A) W3110, W3110∆rsgA and W3110∆rsgA-derived mutant strains, (B) W3110∆rsgA strains
harboring plasmids carrying mutant rbfA and (C) W3110∆rbfA strains harboring plasmids carrying mutant rbfA
are presented.
2
A
B
2
W3110 (pCA24N-)
W3110 (pCA24N-)
W3110∆rsgA (pCA24N-)
W3110 (pCArbfA)
W3110∆rsgA (pCA24N-)
4
W3110∆rsgA (pCArbfA)
A260
OD600
W3110∆rsgA (pCArbfA)
1
2
0
0
0
2
4
6
8
12
Bottom
Time (hour)
C
W3110 (pCA24N-)
W3110 (pCArbfA)
W3110∆rsgA (pCA24N-)
W3110∆rsgA (pCArbfA)
OD600
2
1
0
0
4
Time (hour)
Supplementary Figure 3: Overexpression of RbfA does not restore cell growth and ribosome profile of
W3110∆rsgA.
(A) Mild overexpression of RbfA from pCArbfA does not restore cell growth of W3110∆rsgA.
Growths of W3110 harboring pCA24N- (empty vector), W3110 harboring pCArbfA, W3110∆rsgA harboring
pCA24N- and W3110∆rsgA harboring pCArbfA are presented. 1/100 volume of overnight culture of each strain
was inoculated into prewarmed LB medium containing 10 µg/mL chloramphenicol and cultured at 37 °C.
(B) Mild overexpression of RbfA from pCArbfA does not restore the ribosome profile of W3110∆rsgA.
Ribosome profiles of W3110 harboring pCA24N- (empty vector), W3110∆rbfA harboring pCA24N- and
W3110∆rbfA harboring pCArbfA are presented.
Cells grown in LB medium containing 10 µg/mL
chloramphenicol at 37 °C were harvested when OD600 reached 0.5. The lysate of 10 mg (wet weight) of cells
was subjected to sucrose density gradient centrifugation. The direction of sedimentation is right to left.
(C) High level of overexpression of RbfA from pCArbfA by the addition of IPTG arrests growth of W3110∆rsgA.
Growths of W3110 harboring pCA24N-, W3110 harboring pCArbfA, W3110∆rsgA harboring pCA24N- and
W3110∆rsgA harboring pCArbfA are presented. 1/100 volume of overnight culture of each strain was inoculated
into prewarmed LB medium containing 10 µg/mL chloramphenicol and 1mM IPTG and cultured at 37 °C.
3
Top
(Nucleotide)
1 mM
100 µM
10 µM
1 µM
GTP ATP GTP ATP GTP ATP GTP ATP
Supplementary Figure 4: ATP requires a higher concentration than GTP does for RsgA-dependent release of
RbfA from the 30S subunit.
The 30S subunits (50 nM) with an excess of RbfA (500 nM) were incubated for 30 min at 37 °C and filtered through
YM-100. The complex of RbfA and the 30S subunit that remained on the filter was incubated for 30 min at 37 °C
with 500 nM of wild-type RsgA in the presence of indicated concentrations of GTP or ATP. After incubation,
RbfA that remained on the 30S subunits was obtained by filtration using YM-100, followed by precipitation with
acetone. The precipitate was separated by SDS-PAGE and RbfA was immunochemically detected.
A
1
2
17S
16S
C
B
1
40
mature
immature
with mature 30S ribosomes
with immature 30S ribosomes
without ribosomes
GTP hydrolyzed (µM)
0.8
A260
0.6
0.4
30
20
10
0.2
0
Bottom
0
Top
0
30
60
Time (min)
90
120
Supplementary Figure 5: Characterization of mature and immature 30S ribosomal subunits used in the
experiment for which results are shown in Figure 5.
(A) RNA compositions.
RNA extracted from mature (lane 1) and immature (lane 2) 30S subunits prepared from 70S and 30S fractions,
respectively, of W3110∆rsgA∆rbfA cells was analyzed by 1.5% agarose gel electrophoresis.
(B) Sedimentation profile.
0.56 A260 units of mature and immature 30S subunits were subjected to sucrose density gradient centrifugation.
The direction of the sedimentation is right to left.
(C) Enhancement of the GTPase activity of RsgA by mature or immature 30S ribosome.
Hydrolysis of GTP (50 µM) by RsgA (250 nM) with or without mature or immature 30S subunits (1.4 A260
units/mL) at 37 °C was monitored in 50mM Tris-HCl (pH 7.5), 200 mM KCl, 5 mM MgCl2 and 1 mM DTT. The
level of GTP hydrolysis was quantified by measuring the level of released phosphates using BIOMOL GREEN TM
reagent (BIOMOL).
4
Phylum
Aquificales
Thermotogae
CyanoBacteria
Deinococcus-Thermus
Fusobacteria
Clamydiae
Spirochaetes
Actinobacteria
Firmicutes
Proteobacteria
Source of genome sequence
Aquifex aeolicus
Thermotoga maritima
Nostoc sp. PCC 7120
Synechocystis
Deinococcus radiodurans
Fusobacterium nucleatum
Chlamydia trachomatis
Chlamydophila pneumoniae CWL029
Treponema pallidum
Borrelia burgdorferi
Corynebacterium glutamicum
Mycobacterium tuberculosis H37Rv
Mycobacterium tuberculosis CDC1551
Mycobacterium leprae
Clostridium acetobutylicum
Staphylococcus aureus N315
Listeria innocua
Bacillus subtilis
Bacillus halodurans
Lactococcus lactis
Streptococcus pyogenes M1 GAS
Streptococcus pneumoniae TIGR4
Ureaplasma urealyticum
Mycoplasma pulmonis
Mycoplasma pneumoniae
Mycoplasma genitalium
Pseudomonas aeruginosa
Escherichia coli K12
Escherichia coli O157:H7 EDL933
Escherichia coli O157:H7
Yersinia pestis
Buchnera sp. APS
Xylella fastidiosa 9a5c
Salmonella typhimurium LT2
Vibrio cholerae
Haemophilus influenzae
Pasteurella multocida
Ralstonia solanacearum
Neisseria meningitidis MC58
Neisseria meningitidis Z2491
Helicobacter pylori 26695
Helicobacter pylori J99
Campylobacter jejuni
Caulobacter vibrioides
Agrobacterium tumefaciens strain C58 (Cereon)
Sinorhizobium meliloti
Brucella melitensis
Mesorhizobium loti
Rickettsia prowazekii
Rickettsia conorii
COG0858
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
COG1162
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Supplementary Table I: Phylogenetic distributions of COG0858 (orthologs of E. coli RsgA) and COG1162
(orthologs of E. coli RbfA) in bacterial entries of the COG database (Tatusov et al, 1997, 2003).
“+” and “-” represent the presence and absence of the gene for COG0858 or COG1162 in the bacterial genome,
respectively.
5
References
Huang YJ, Swapna GV, Rajan PK, Ke H, Xia B, Shukla K, Inouye M, Montelione GT (2003) Solution NMR
structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli. J Mol
Biol 327: 521–536
Kraulis P (1991) MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures J
Appl Cryst 24: 946–950
Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R,
Mekhedov SL, Nikolskaya AN, Rao BS, Smirnov S, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA
(2003) The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4: 41
Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein families. Science 278: 631–637
6