See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/46149753

A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation

responses

Article  in  Nature Structural & Molecular Biology · October 2010

DOI: 10.1038/nsmb.1906 · Source: PubMed

CITATIONS READS
49 253

15 authors, including:

Dawei Sun Jun Hee Lee

Paul Scherrer Institut Dong-A University
10 PUBLICATIONS   364 CITATIONS    271 PUBLICATIONS   5,809 CITATIONS   

SEE PROFILE SEE PROFILE

Hye-Yeon Kim Kyung-Jin Kim

Korea Basic Science Institute KBSI Ewha Womans University
37 PUBLICATIONS   474 CITATIONS    148 PUBLICATIONS   1,822 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

N-end rule pathway View project

cancer, centrosome View project

All content following this page was uploaded by Dawei Sun on 10 April 2014.

The user has requested enhancement of the downloaded file.

 articles

A metazoan ortholog of SpoT hydrolyzes ppGpp and

functions in starvation responses
Dawei Sun1,2,10, Gina Lee3,4,10, Jun Hee Lee3,10, Hye-Yeon Kim1, Hyun-Woo Rhee5, Seung-Yeol Park3,
Kyung-Jin Kim6, Yongsung Kim3,4, Bo Yeon Kim7, Jong-In Hong5, Chankyu Park3, Hyon E Choy8,
Jung Hoe Kim3, Young Ho Jeon1,2 & Jongkyeong Chung3,4,9

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses.
© 2010 Nature America, Inc. All rights reserved.

Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in
Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain
an active site for ppGpp hydrolysis and a conserved His-Asp–box motif for Mn2+ binding. Consistent with these structural
data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3′,5′-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also
suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9)
in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino
acid–starved Mesh1 null mutant has highly downregulated DNA and protein synthesis–related genes and upregulated
stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in
starvation responses.

Upon amino acid starvation, bacteria slow down the physiological
processes for cell growth and speed up the processes to overcome
nutrient deficit. This stringent response is regulated by the RelA and
SpoT proteins, which catalyze synthesis and degradation of a signal-
ing alarmone, ppGpp1–3 (Fig. 1a). To produce ppGpp, RelA cata-
lyzes pyrophosphorylation of GDP (or GTP) using ATP2–4 (Fig. 1a).
Conversely, SpoT, an Mn2+-dependent pyrophosphohydrolase, specifi-
cally hydrolyzes ppGpp into GDP and pyrophosphate (PPi)2,3 (Fig. 1a).
SpoT can also synthesize ppGpp (Fig. 1a) under carbon or fatty acid
limitation conditions2,3,5. As ppGpp is required for the stringent
responses, double deletion mutants for relA and spoT are lethal in
minimal media6,7. In contrast, spoT hypomorphic mutants or moder-
ately RelA-expressing bacteria show delayed growth because increased
intracellular ppGpp suppresses cell growth8,9. spoT null mutants are
also lethal, as an uncontrolled large increase in ppGpp level severely
inhibits cell growth7–9.
RelA and SpoT proteins were initially identified in Escherichia coli,
but the function of these enzymes is largely conserved in most
bacteria 2,3, suggesting that the stringent response mechanism
mediated by RelA and SpoT has conferred a selective growth advan-
tage in prokaryotes. A growing number of reports show that RelA
and SpoT proteins are even conserved in plants, including the
unicellular green alga Chlamydomonas reinhardtii 10 and higher
plants such as Nicotiana tabacum11, Arabidopsis thaliana12–14 and
rice15. Like their bacterial homologs, these plant RelA and SpoT
proteins catalyze synthesis and degradation of ppGpp and com-
plement the growth defects of E. coli relA and spoT mutants10–15.
Notably, most plant RelA and SpoT homologs contain chloroplast
targeting motifs in their N-terminal sequences and thus are thought
to originate from lateral gene transfer of endosymbiotic cyanobac-
teria11,15,16. Indeed, these plant genes are mainly expressed in green
tissues (that is, in tissues with chloroplasts), and their expression
changes dynamically during plant development13–15. Furthermore,
the knockdown mutation of an Arabidopsis RelA and SpoT homolog
(RSH) causes developmental defects, including fertilization and
reproduction problems13, highlighting the physiological importance
of RelA and SpoT homologs in plants.
However, until now, no RelA or SpoT homologs have been identi-
fied in animals17,18. Here we provide the first evidence that func-
tional SpoT homologs exist in animals—Drosophila melanogaster
and human. These metazoan SpoT homologs have structural and
functional properties very similar to those of bacterial ppGpp
hydrolases. Furthermore, the null mutant for the Drosophila SpoT
homolog shows retarded body growth and impaired starvation

1Division of Magnetic Resonance, Korea Basic Science Institute, Chungbuk, South Korea. 2Department of Bio-Analytical Science, University of Science and
Technology, Daejeon, South Korea. 3Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea. 4National
Creative Research Initiatives Center and School of Biological Sciences, Seoul National University, Seoul, South Korea. 5Department of Chemistry, Seoul National
University, Seoul, South Korea. 6Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, South Korea. 7Korea Research Institute of
Bioscience and Biotechnology, Chungbuk, South Korea. 8Department of Microbiology, Chonnam National University Medical College, Kwangju, South Korea.
9Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea. 10These authors contributed equally to this work. Correspondence should

be addressed to Y.H.J. (yhjeon@kbsi.re.kr) or J.C. (jkc@snu.ac.kr).

Received 1 October 2009; accepted 13 July 2010; published online 5 September 2010; doi:10.1038/nsmb.1906

1188 VOLUME 17  NUMBER 10  OCTOBER 2010  nature structural & molecular biology
 articles

Figure 1  Identification of SpoT homologs in

a b Human HD 179 residues

Animals
Drosophila HD 179 residues
metazoa. (a) Schematic diagram showing RelA
GDP
ATP PPi C. elegans HD 229 residues
and SpoT enzymes catalyzing the synthesis and
Arabidopsis-c HD tSD 216 residues
degradation of ppGpp. ppGpp can be converted
from guanosine 3′-diphosphate 5′-triphosphate

Plants
ReIA SpoT Arabidopsis-b Chl HD SD 710 residues
Arabidopsis-a Chl HD SD RD1 RD2 883 residues
(pppGpp)2. Modified from ref. 2. (b) Comparison
SpoT
Synechocystis sp. HD SD RD1 RD2 760 residues
of the domain structures of the ppGpp hydrolase

Bacteria
AMP Streptococcus equisimilis HD SD RD1 RD2 739 residues
domain–containing SpoT homologs from various
ppGpp species. HD, hydrolase domain; SD, synthetase
E. coli HD SD RD1 RD2 702 residues
Hydrolase Synthetase Regulatory Regulatory domain; tSD, truncated synthetase domain; RD,
domain domain domain 1 domain 2 regulatory domain; Chl, chloroplast targeting
c Human MESH1
Time (min)
Drosophila Mesh1
Time (min)
d motif. Stripes indicate inactive synthetase
domains. For more details, see Supplementary
400 400

h1
0 0

H
Figure 1. (c,d) Chemosensor PyDPA (c)
Intensity (AU)
Intensity (AU)

ES
ST

ST

es
300 0.5 300 0.5

M
G

G
1 1 and TLC (d) analyses showing the ppGpp
200 3 200 3
5 5 hydrolysis activity of human MESH1 (left) and
100 100
7 7 Drosophila Mesh1 (right) purified from E. coli (c),
0 0
350 425 500 575 650 350 425 500 575 650 HEK293T (d, left) or Drosophila S2 cells
Wavelength (nm) Wavelength (nm) (d, right). The fluorescence intensities (AU,
e α7
arbitrary units) at different times are plotted in c.
N α10 α6
In d, white and black arrowheads indicate
α5 locations of PPi and ppGpp, respectively, on TLC
α3 C 90°
α1
N α3 α4 α8
α9 plates. All assays were performed with 0.5 μg
© 2010 Nature America, Inc. All rights reserved.

α5 α2 α8 C of purified Mesh1 proteins. (e) Side (left) and

α6 top (right) views of human MESH1 structures
β1 Mn2+
α9
Mn2+ α1 represented as ribbon diagrams. N-terminal,
α4 α7 α2 C-terminal and central α-helices are colored
β2 β2
α10 red, blue and cyan, respectively. Green sheets,
β1
β-hairpins (β1-β2); magenta sphere, Mn2+
ion. (f,g) Superimpositions of human MESH1
f N g (orange) and Drosophila Mesh1 (green) (f), or of
MESH1 and the hydrolase domain of bacterial
N
N C RelSeq (gray) with ppG2′:3′p (ref. 28; g).
C
α-helices are represented as cylinders. Mn2+
C
ions in MESH1 and RelSeq are shown as
Mn2+
magenta and gray spheres, respectively.
Mn2+ N C
Consequently, the amount of hydrolyzed
ppG2′:3′p
GDP and PPi increased after Mesh1 addition,
as determined by fluorescence intensity at
380 nm20 (Fig. 1c). We then purified recom-
responses, suggesting that the metazoan SpoT homologs are crucial binant MESH1 and Mesh1 proteins from human HEK293T and
in animal physiology, as they are in bacteria and plants. Drosophila S2 cells, respectively. Like those purified from E. coli, these
purified proteins efficiently induced ppGpp hydrolysis, as shown by
RESULTS the concomitant disappearance and appearance of equivalent amounts
Identification of the metazoan SpoT ortholog Mesh1 of ppGpp and PPi, respectively, in TLC analyses (Fig. 1d)11.
To identify putative metazoan SpoT, we searched for homologs of To examine whether the hydrolase activity of Mesh1 is specific to
E. coli SpoT in human and various animals such as mouse, zebrafish, ppGpp, we used HPLC to measure its turnover of potential substrates
Xenopus laevis, Drosophila and Caenorhabditis elegans. We identified of phosphate hydrolases, including ATP, GTP, dATP, dGTP, cyclic AMP,
a single ortholog in each organism, all of which contain a highly cons­ cyclic GMP, dAMP, dGMP and AppppA (Ap4A)22,23. Notably, only
erved His-Asp–box motif, required for ppGpp hydrolase activity18,19 ppGpp, and no other nucleotide, was effectively hydrolyzed by human
(Fig. 1b and Supplementary Fig. 1). The metazoan SpoT homologs MESH1 (Supplementary Table 1 and Supplementary Fig. 2). These
(which we named metazoan SpoT homolog-1, or Mesh1) consist only results show that Mesh1 has a specific ppGpp hydrolase activity.
of the hydrolase domain of bacterial SpoT and lack the synthetase and
regulatory domains3,18. Mesh1 has a similar structure to bacterial ppGpp hydrolase
To examine the biochemical properties of these potential SpoT We next determined the crystal structures of the Mesh1 proteins.
homologs, we cloned full-length human MESH1 (HDDC3) and Table 1 summarizes the data processing and refinement statistics.
Drosophila Mesh1, and purified the recombinant proteins MESH1 Overall, human MESH1 and Drosophila Mesh1 have highly similar
and Mesh1 from E. coli. To measure their ppGpp hydrolysis acti­vity, structures, composed of ten α-helices and two β-strands that con-
we used the chemosensor pyrene and bis(Zn2+-dipicolylamine) stitute three subdomains (Fig. 1e,f and Supplementary Fig. 3). The
(PyDPA), which generates fluorescence at 470 nm when it speci­ N-terminal subdomain of the Mesh1 proteins contains an antiparallel
fically binds to ppGpp20. We also used thin-layer chromatography helix bundle including helices α1 to α3 (Fig. 1e). An extended loop
(TLC) to detect radioactive ppGpp (3′-[β-32P]ppGpp)1,11. Addition between α1 and α2 forms a short β-hairpin (β1-β2; Fig. 1e). The
of 0.5 μg of purified human MESH1 or fly Mesh1 protein strongly C-terminal subdomain containing α7 to α10 consists of an antiparallel
induced ppGpp hydrolysis within 5 min in a time-dependent manner four-helix bundle folded by two helix-turn-helix motifs (Fig. 1e).
(Fig. 1c), ana­logous to the kinetics of ppGpp hydrolysis in E. coli21. The central region between the N- and C-terminal subdomains has

nature structural & molecular biology  VOLUME 17  NUMBER 10  OCTOBER 2010 1189
 articles

Table 1  Structure data collection, phasing and refinement and Supplementary Table 2)27,29. These data demonstrate that
statistics Mesh1’s enzymatic activity for ppGpp hydrolysis is comparable to
Native human SeMet human Native Drosophila that of the bacterial enzyme.
MESH1 MESH1 Mesh1

Data collection Crucial residues for the ppGpp hydrolysis activity of Mesh1
Space group P21 P21 P21 A detailed view of the superimposition of MESH1 and RelSeq revealed
Cell dimensions that all the key residues for ppGpp hydrolysis in RelSeq28 are similarly
  a, b, c (Å) 53.9, 62.4, 53.9 52.7, 63.0, 52.7 50.0, 71.6, 49.9 projected into the active site of MESH1, including Arg24, Glu65, Asp66
  α, β, γ (°) 90.0, 95.3, 90.0 90.0, 93.4, 90.0 90.0, 101.3, 90.0 and Asn126 (Fig. 2b). To examine the importance of these conserved
Wavelength (Å) 0.9795 0.9796 1.2398 catalytic residues, we mutated each residue and measured mutants’
Resolution (Å) 50–1.9 50–2.0 50–2.9 ppGpp hydrolysis activities using PyDPA. Like the bacterial RelSeq
(1.97–1.90) (2.12–2.00) (3.0–2.9) mutations28, R24A and E65A mutations in human MESH1 (R25A and
Rsym 0.069 (0.308) 0.074 (0.31) 0.071 (0.366) E66A in Drosophila Mesh1) mostly abrogated ppGpp hydrolase activity,
I / σI 15.8 (2.2) 29.8 (4.9) 20.2 (2.2) and a D66A mutation in human MESH1 (D67A in Drosophila Mesh1)
Completeness (%) 96.3 (93.3) 99.7 (97.5) 96.5 (80.4) substantially reduced the hydrolase activity (Fig. 2c,d). These results
Redundancy 2.0 (1.9) 7.4 (6.5) 3.5 (2.7) imply that the ppGpp hydrolysis site is both structurally and function-
ally conserved between Mesh1 and bacterial SpoT.
Refinement
Notably, one Mn2+ ion, which in bacteria is known to couple with
Resolution (Å) 50–1.9 20–2.9
ppGpp and activate ppGpp hydrolysis3,28, is coordinated with His35,
No. reflections 27,212 7,066
His61, Asp62 and Asp122 in human MESH1 (Fig. 2e; His36, His62,
© 2010 Nature America, Inc. All rights reserved.

Rwork / Rfree 0.203 / 0.230 0.213 / 0.282

Asp63 and Asp123 in Drosophila Mesh1). To determine whether Mn2+ is
No. atoms
essential for ppGpp hydrolase activity in animal Mesh1, as it is in bacte-
  Protein 2,850 2,870
rial homologs, we incubated radioactive ppGpp with Drosophila Mesh1
  Mn2+ 2 2
in Mn2+ or Mg2+ buffer. Mesh1 in Mn2+ buffer efficiently hydro-
  SO42− – 10
lyzed ppGpp in a time-dependent manner (Fig. 2f, left), whereas
  Water 220 36
ppGpp hydrolysis was much less efficient in Mg2+ buffer (Fig. 2f,
B-factors
right). Moreover, mutation of His62 in Drosophila Mesh1, which lies
  Protein 23.49 66.22
in the His-Asp–box motif and coordinates Mn2+ ion, to phenyl­alanine
  Mn2+ 28.98 52.23
(Mesh1 HF) completely disrupted the ppGpp hydrolase ­ activity
  SO42− – 81.53
(Fig. 2g). These data indicate that Mesh1 has a Mn2+-dependent
  Water 36.06 39.02
ppGpp hydrolase activity similar to that of bacterial SpoT.
R.m.s. deviations
  Bond lengths (Å) 0.005 0.008
Mesh1 catalyzes ppGpp hydrolysis in vivo
  Bond angles (°) 1.0 1.2
We next investigated whether Mesh1 proteins act as functional
One crystal was used for each data set. Values in parentheses are for highest-resolution
shell. SeMet, selenomethionyl. ppGpp hydrolases in vivo. We expressed E. coli RelA in HEK293T
cells and cultured them in the presence of [32P]H 3PO 4 to induce
synthesis of radioactive ppGpp. Notably, expression of wild-type
three short α helices (α4 to α6; Fig. 1e). The helical turn between α3 MESH1 (MESH1 WT) completely prevented ppGpp accumula-
and α4 contains a conserved His-Asp–box motif (Supplementary tion induced by RelA (Fig. 3a). However, MESH1 HF did not
Fig. 3)—the motif found in the superfamily of metal-dependent show this inhibitory effect (Fig. 3a). To examine whether Mesh1
phosphohydrolases18,19—and coordinates one Mn2+ ion together proteins can also hydrolyze ppGpp in E. coli cells, we expressed
with helices α2 and α8 (Fig. 1e). Drosophila Mesh1 WT under the control of the tac promoter in
A search for the structural homologs of MESH1 identified several E. coli and stimulated ppGpp synthesis by amino acid starvation,
His-Asp–box–containing metallophosphohydrolases19,24, including treating cells with serine hydroxamate or a high concentration of
the E. coli 5′-deoxynucleotidase YfbR (PDB 2PAQ (ref. 25); Z-score, 8.1; valine 1,30,31. IPTG-induced expression of Drosophila Mesh1 WT
r.m.s. deviation, 3.7 Å for 126 Cα atoms) and human cAMP-specific reduced the ppGpp pool in a dose-dependent manner (Fig. 3b),
3′,5′-cyclic phosphodiesterase-4 (PDB 1F0J (ref. 26); Z-score, 6.0; whereas this was not observed with Mesh1 HF (Supplementary
r.m.s. deviation, 3.5 Å for 139 Cα atoms). However, the most highly Fig. 4a). These results indicate that Mesh1 proteins can function
conserved protein was the Streptococcus equisimilis RelA and SpoT as ppGpp hydrolases in vivo.
homolog RelSeq27 (PDB 1VJ7 (ref. 28); Z-score, 17.6). Superimposition
of the hydrolase domain of RelSeq with human MESH1 or fly Mesh1 Mesh1 rescues the lethality of SpoT deficiency in E. coli
structures gives r.m.s. deviations of, respectively, 2.1 Å for 151 Cα The hallmark changes in bacteria after accumulation of ppGpp are
atoms and 2.3 Å for 152 Cα atoms. Notably, the active site of ppGpp reductions of stable RNA synthesis and growth-related gene expres-
hydrolysis in RelSeq28 coincides with the proposed active pocket sion, and increases in amino acid synthesis, proteolysis and stress-
formed by β1, α4 and α8 in the Mesh1 proteins (Fig. 1g), suggesting responsible gene expression32–36. Strains lacking ppGpp are nonviable
that Mesh1 and RelSeq share a common active-site fold. in amino acid–deficient minimal media6,7, and bacterial growth is
We next compared the ppGpp hydrolysis activity of Mesh1 with that inhibited by high levels of ppGpp even in nutrient-rich media7–9. We
of RelSeq. The kcat/Km ratios of human MESH1 and Drosophila Mesh1 observed that growth of wild-type E. coli on LB plates as well as in
were 9.46 × 103 and 9.58 × 103 M−1 s−1, respectively, and the kcat/Km liquid media was suppressed by RelA overexpression (Fig. 3c,d) owing
of the RelSeq D264G variant (containing a mutation that abolishes the to high levels of ppGpp (Supplementary Fig. 4b)9. Notably, coexpres-
ppGpp synthetase activity of RelSeq28) was 10.34 × 103 M−1 s−1 (Fig. 2a sion of Drosophila Mesh1 WT, but not Mesh1 HF, strongly suppressed

1190 VOLUME 17  NUMBER 10  OCTOBER 2010  nature structural & molecular biology
 articles

a 14 Human MESH1 b e
12 Drosophila Mesh1
1 / V0 × 10–6 (s M–1)

RelSeqD264G
10
His61
8
6
Mn2+
4 Asp62 Asp122
2
0 His35
−2 0 2 4 6 8 10 12
1 / [ppGpp] × 10–3 (M–1)

c 1.0
Human MESH1 d 1.0
Drosophila Mesh1 f Mesh1 WT
g
0.9 0.9
Mn2+ Mg2+

h1 T
0.8

F
0.8

M 1W

H
0.7 0.7

h
ST

es
es
s

s
m

m
m
m

m
Time

s
15

30

M
G
0.6 0.6

2
4
8
0
8
Ft / F0

0.5 Ft / F0
0.5
ppGpp only ppGpp only
0.4 0.4
R24A R25A
0.3 E65A 0.3
E66A
0.2 D66A 0.2 D67A
0.1 WT 0.1 WT
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min) Time (min)
© 2010 Nature America, Inc. All rights reserved.

Figure 2  Mesh1 compared with bacterial ppGpp hydrolase shows a conserved structure and similar enzymatic activity. (a) ppGpp hydrolysis activity of
human MESH1, Drosophila Mesh1 and bacterial RelSeq D264G using HPLC analyses. The D264G mutation abolishes the ppGpp synthetase activity of
RelSeq28. Error bars indicate s.d. from three independent experiments. See Supplementary Table 2 for the values of kinetic parameters. (b) Stereo view
of superimposition of the active sites of MESH1 (orange) and Rel Seq (gray). Mn2+ ions in MESH1 and RelSeq are shown as magenta and gray spheres,
respectively. ppG2′:3′p bound to RelSeq28 is displayed as gray sticks. (c,d) PyDPA analyses showing ppGpp hydrolysis activity of human MESH1 (c) and
Drosophila Mesh1 (d) proteins. The relative intensity (Ft/F0) is plotted versus time; F0 is the initial fluorescence intensity at 470 nm in the presence
of 5 μM ppGpp; Ft is the fluorescence intensity at 470 nm after addition of Mesh1 protein at the indicated times. All assays were performed with 0.5
μg of purified Mesh1 proteins. Error bars indicate s.d. from three independent experiments. (e) Close-up view of the Mn2+-binding site in MESH1 with
2Fo –Fc map (deep blue) contoured at the 1σ level. Magenta sphere, Mn2+ ion; green sticks, Mn2+-coordinating residues; red spheres, water molecules;
dotted lines, ionic bonds. (f,g) TLC analyses showing ppGpp hydrolysis activities of the indicated Mesh1 proteins with Mn 2+ or Mg2+. White arrowheads,
PPi; black arrowheads, ppGpp; m, minutes.

the RelA-induced growth retardation (Fig. 3c,d), which suggests that overcome amino acid limitation7. Collectively, these results indicate
Mesh1 inhibits ppGpp accumulation in E. coli. Furthermore, expres- that Mesh1 efficiently hydrolyses ppGpp in E. coli.
sion of Drosophila Mesh1 WT, but not Mesh1 HF, prevented E. coli We next examined whether Mesh1 can complement the endogenous
growth in minimal media (Supplementary Fig. 4c), which may be role of bacterial SpoT. As previously reported7,8, we found that ΔrelA
because it lowers ppGpp abundance to below the level required to ΔspoT double-mutant E. coli grew well in complete media (Fig. 3e,f).

a b c pG184
pGEX
pG184-relA
pGEX
pG184-relA
pGEX-Mesh1 WT
pG184-relA
pGEX-Mesh1 HF
H T
F
ES W

GST Mesh1
1

1
H
ES
ST

ST

IPTG
M

M
G

0 0 0.01 0.1 1 (mM)

RelA – + + +

e �relA �spoT f
OO
IB: Mesh1 OO
R
T

d
M
W

pG184 + pGEX pG184 + pGEX

pG184-relA + pGEX pG184-relA + pGEX
10 10
WT

RM

pG184-relA + pGEX-Mesh1 WT pG184-relA + pGEX-Mesh1 WT

pG184-relA + pGEX-Mesh1 HF pG184-relA + pGEX-Mesh1 HF
pG184 pG184-relA LB complete medium
1 1 pGEX pGEX
OO
OO
A600

A600

R
T

0.1 0.1
M
W
WT

RM

LB only LB + 0.05 mM IPTG

0.01 0.01
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 pG184-relA pG184-relA M9 minimal medium
Time (h) Time (h) pGEX-Mesh1 WT pGEX-Mesh1 HF

Figure 3  Mesh1 hydrolyzes ppGpp in vivo. (a,b) TLC analyses of ppGpp hydrolase activities of Mesh1 proteins expressed in HEK293T (a) or E. coli (b)
cells. ppGpp synthesis was induced by RelA expression (a) or serine hydroxamate treatment (b). IPTG was used to induce Mesh1 WT expression (b),
which was confirmed by immunoblot (IB) analyses (b, bottom gel). Gray arrowhead, GTP; black arrowheads, ppGpp. (c) E. coli (DH10β) cells transformed
with the indicated plasmids were plated, exposed to filter disks soaked in 5 mM (top left), 2 mM (top right), 1 mM (bottom left) and 0.5 mM (bottom right)
of IPTG and incubated for 12 h to visualize the growth-inhibition zones surrounding each disk. (d) The E. coli cells used in c were grown in liquid media
without (left) or with IPTG (right). (e) ΔrelA ΔspoT E. coli (CF1693) cells transformed with the indicated plasmids were plated and grown for 12 h.
(f) Wild-type E. coli cells (CF1648; WT) or ΔrelA ΔspoT (CF1693) E. coli cells transformed with pG184 and pGEX (OO) or pG184-relA and pGEX-Mesh1
WT (RM) plasmids were streaked on media and grown for 12 h.

nature structural & molecular biology  VOLUME 17  NUMBER 10  OCTOBER 2010 1191
 articles

a b c WT 5A3
d e WT 5A3
Size
(BgIII) 50
(kbp) Mesh1 G3858
10 40

IB: Mesh1
3.9
100 bp WT 5A3 30
CG14508 Mesh1 3.0
2.0
1.6 Mesh1
1.0 20
0.5 Actin Size (kDa)
0.1 CG11899
28S Mesh1 5A3 Size (kbp) IB: Tub
rRNA BgIII BgIII
E L P F M
f 80
WT
5A3
g h WT
5A3
Figure 4  Mesh1 null mutant da > relA
Eclosion rate (%)
48 h 100
Drosophila shows retarded body 60 5A3; da > Mesh1
80

Viability (%)
growth and impaired starvation 40
60
resistance. (a) Developmental
northern blot analyses of Mesh1. 20 120 h 40
(AEL)
28S rRNA was used as a loading 0 20
control. E, embryo; L, larva; 8 9 10 11 12 13 14 0
Time AEL (d) WT 5A3 da > relA 5A3; da > Day 1 Day 3 Day 5
P, pupa; F, adult female; M, adult Time after starvation
Mesh1
male. (b) Schematic representation
of Mesh1 genomic locus and the i WT 5A3
CycE
j
genomic deletion region of Mesh1 BrdU 50% Hoechst 53%

5A3. (c–e) Southern blot (c), DNApol

© 2010 Nature America, Inc. All rights reserved.

RT-PCR (d) and immunoblot (e) Pole2

Fed

analyses of wild-type (WT) and

hoip
Mesh1 5A3 mutant (5A3). Actin (d)
and tubulin (Tub; e) were used as RnrL
loading controls. (f–h) Experiments 21% 1%
Hsp26
comparing adult eclosion rate (f),
larval development (g) and viability Hsp70A
Stv

in an amino acid–starved media (h)

Actin
among WT, Mesh1 5A3, da > relA
Fed Stv Fed Stv Fed Stv
and Mesh1 5A3; da > Mesh1 flies
WT 5A3 da > relA
at the indicated times after egg
laying (AEL; f,g) or after starvation (h).
Scale bar, 1 mm. For more detailed information about Drosophila genotypes, see Supplementary Methods. (i) Immunostaining with BrdU-specific
antibody (green) and Hoechst 33258 staining (DNA, blue) in WT and Mesh1 5A3 larval fat bodies, which were fed (top) or amino acid–starved
(Stv, bottom) for 20 h. Numbers indicate the percentage of BrdU-labeled cells (n ≥ 5). Scale bar, 50 μm. (j) RT-PCR analyses for the indicated genes
in WT, Mesh1 5A3 and da > relA larvae which were fed or amino acid–starved (Stv) for 8 h. Actin was used as a loading control.

RelA expression in this genetic background made them less viable
(Fig. 3e) owing to excessive accumulation of ppGpp in the absence of
the hydrolytic enzyme SpoT7,8. Notably, expression of Drosophila Mesh1
WT, but not Mesh1 HF, was sufficient to suppress this lethality (Fig. 3e,f).
Moreover, these relA+ΔspoT Mesh1 transformants also grew well on
minimal medium (Fig. 3f). These data strongly support an evolutionarily
conserved ppGpp hydrolase function of Mesh1 and E. coli SpoT.
Mesh1 deletion impairs starvation resistance in Drosophila
Northern blot analyses in Drosophila indicated that Mesh1 is highly
expressed in the larval stage (Fig. 4a), in which considerable food uptake
and body growth occurs. By imprecise excision of a P-element from
the Drosophila Mesh1 G3858 allele, we generated a Drosophila Mesh1
null allele, Mesh1 5A3 (Fig. 4b), which we confirmed by Southern
blot, reverse-transcription PCR (RT-PCR) and immunoblot analyses
(Fig. 4c–e). Mesh1-deficient flies were viable but showed retarded body
growth, which was rescued by transgenic Mesh1 expression (Fig. 4f,g
and Supplementary Fig. 5). Furthermore, the Mesh1 null mutant was
much more susceptible than wild-type flies to death from amino acid
starvation, and this susceptibility was also completely rescued by Mesh1
expression (Fig. 4h). These results indicate that Mesh1 is important in
body growth and starvation resistance in Drosophila.
Mesh1 null mutant shows altered gene expression profiles
To further explore the effect of Mesh1 deficiency on cell growth, we
performed bromodeoxyuridine (BrdU) incorporation assays in the
Drosophila larval fat body37. In a ‘fed’ condition, we detected compa-
rable BrdU incorporation in Mesh1 null mutant and wild-type flies
(Fig. 4i). On the other hand, the amino acid–starved Mesh1 null
mutant showed much less BrdU incorporation than the wild type
(Fig. 4i). Consistent with these results, genome-wide microarray ana­
lyses indicated that expression of genes associated with DNA and
protein synthesis was markedly reduced in starved Mesh1 null mutants
(Table 2, Supplementary Table 3a and Supplementary Methods;
categorized by Gene Ontology38,39). In contrast, expression of stress-
responsive genes was much higher in starved Mesh1 null mutants
(Table 2 and Supplementary Table 3b). The gene categories altered in
Mesh1 null mutants as a result of starvation were quite different from
those in wild-type flies—for example, starved wild-type flies showed
substantial downregulation of the genes for lipid and carbohydrate
metabolism (Supplementary Table 3c)39,40.
We also confirmed the expression levels of the affected genes using
RT-PCR. Again, genes related to DNA replication, including CyclinE,
DNApol and Pole2, as well as those related to protein translation,
such as hoip and RnrL, were highly downregulated in the starved
Mesh1 null mutant, whereas genes related to stress responses, includ-
ing Hsp26 and Hsp70A, were highly upregulated (Fig. 4j). According
to reports, regulation by RelA produces similar gene expression pat-
terns in response to amino acid starvation in bacteria34–36. Therefore,
we expressed E. coli RelA in Drosophila (Supplementary Fig. 5) and
examined its effect. RelA-expressing flies showed severe develop-
mental delay (Fig. 4g) and impaired starvation resistance (Fig. 4h),

1192 VOLUME 17  NUMBER 10  OCTOBER 2010  nature structural & molecular biology

Table 2  Affected gene categories in Drosophila Mesh1 null mutant
in response to amino acid starvation
GO category Number of genes (total) P of ppGpp (detection limits of our systems were around 500 pmol for
Downregulated genes
Ribonucleoprotein complex 63 (391) 1.11 × 10−14
Mitochondrial ribosome 27 (77) 1.66 × 10−14
RNA processing 58 (357) 1.58 × 10−12
Ribosome biogenesis and assembly 22 (55) 3.61 × 10−12
DNA replication 35 (159) 4.07 × 10−12
rRNA metabolic process 18 (44) 5.43 × 10−10
tRNA metabolic process 20 (82) 3.38 × 10−07
Upregulated genes
Response to biotic stimulus 18 (167) 5.23 × 10−08
Defense response 24 (324) 7.80 × 10−07
Endopeptidase inhibitor activity 14 (104) 7.75 × 10−06
Response to unfolded protein 6 (25) 1.35 × 10−04
The Gene Ontology (GO) categories of differentially regulated genes are listed. GO sta-
tistical analyses were performed for genes downregulated or upregulated in response to
amino acid starvation in Mesh1-null flies. For each GO category, the number of affected
genes and the P value of the match are indicated. The total number of genes in each
© 2010 Nature America, Inc. All rights reserved.
GO category is indicated in parentheses. See Supplementary Table 3 for more details.
which were very similar to Mesh1 null mutant phenotypes (Fig. 4g,h).
Furthermore, most of the genes with altered expression in Mesh1 null
mutant were also similarly regulated in RelA-expressing flies (Fig. 4j).
Collectively, these data indicate that the evolutionarily conserved
function of Mesh1 ppGpp hydrolase is to regulate gene expression
related to growth and stress responses in Drosophila.
DISCUSSION
Bacterial SpoT has both hydrolase and synthetase domains and is
able to synthesize ppGpp in response to stresses such as carbon
and fatty acid limitation3,5. In contrast, the metazoan enzyme
Mesh1 has only a ppGpp hydrolase domain (Fig. 1b) and does not
synthesize ppGpp (Supplementary Fig. 4b). BLAST search analyses
using amino acid sequences of SpoT domains (the hydrolase,
synthetase and regulatory domains) revealed that most ppGpp
hydrolase domain–containing proteins in bacteria and plants have all
three domains, just like SpoT in E. coli (Fig. 1b and Supplementary
Fig. 1)3,11–16. We also found proteins in plants containing only
a hydrolase domain and a truncated synthetase domain (Fig. 1b and
Supplementary Fig. 1). In contrast, all animal species we searched
(including C. elegans, Drosophila, zebrafish, Xenopus, mouse and
human) have only hydrolase domain–containing proteins like Mesh1
(Fig. 1b and Supplementary Fig. 1), suggesting that synthetase and
regulatory domains have been lost or altered in these species. These
results show that Mesh1 has diverged greatly from SpoT during the
evolution of animals.
Therefore, it is possible that other proteins with unrelated sequences
have evolved to function as ppGpp synthetases or regulators of Mesh1,
as suggested by some plant RelA and SpoT homologs whose regula-
tory-domain sequences are entirely different from those of bacte-
rial homologs14,15. Notably, several enzymes capable of producing
ppGpp, such as 3′-OH pyrophosphoryl transferase and nucleotide
2′,3′-cyclic monophosphokinase, have amino acid sequences com-
pletely unrelated to common ppGpp synthetases3,41–44. Moreover,
the three-dimensional structures of eukaryotic nucleotidyltransferase
superfamily proteins (E.C.2.7.7.-) are highly similar to those of bacte-
rial ppGpp synthetases—the similarity is comparable to that among
the nucleotidyltransferases themselves—even though the overall
amino acid sequences are not closely related28,45.
nature structural & molecular biology  VOLUME 17  NUMBER 10  OCTOBER 2010 1193
articles
We attempted to measure endogenous ppGpp in Drosophila using
HPLC and MALDI-TOF MS, but we did not find a detectable amount
HPLC and 10 pmol for MS) when analyzing up to 160 mg (for HPLC)
or 4 mg (for MS) of amino acid–starved Mesh1-null Drosophila larvae.
This suggests that the ppGpp level in Drosophila is below 2,500 pmol g−1,
which is less than ppGpp levels in amino acid–starved bacteria
(5,000–50,000 pmol g−1)46,47 but may be comparable to ppGpp levels
in wounded plant chloroplasts (up to 1,874 pmol g −1)22. Our result
is also consistent with previous evidence that there is less than 1,000
pmol g−1 ppGpp in animal cells17,48,49.
Nevertheless, our results strongly suggest that the previously
uncharacterized metazoan SpoT homolog Mesh1 has ppGpp hydroly-
sis activity and is involved in starvation responses in vivo. First, Mesh1
has an active site structure for ppGpp hydrolysis, and the crucial resi-
dues for the ppGpp hydrolysis in bacterial SpoT are conserved in
Mesh1 (Figs. 1 and 2). Second, the hydrolase activity of Mesh1 is
highly specific to ppGpp among various nucleotides and is compara-
ble to that of bacterial RelSeq (Supplementary Tables 1 and 2). Mesh1
catalyzes ppGpp hydrolysis in vivo as well as in vitro and genetically
complements SpoT deficiency in E. coli (Fig. 3). Finally, deletion of
Mesh1 in Drosophila causes developmental delay and impaired starva-
tion resistance (Fig. 4).
The categories of genes whose expressions are altered in amino
acid–starved Mesh1-null flies (Table 2 and Supplementary Table 3)
are highly similar to those regulated by RelA and SpoT in bacteria
(that is, genes related to cell growth and stress responses)34–36.
Notably, we found that genes associated with endoplasmic reticulum
homeostasis and mitochondrial ribosome proteins are downregulated
in Mesh1-null flies, and genes related to innate immune responses
are upregulated (Supplementary Table 3); these gene categories are
irrelevant in bacteria. These results imply that gene regulation and
control of cellular processes by SpoT has been not only evolutionarily
conserved but also further developed to support more complex physio­
logy in higher organisms (Supplementary Fig. 6). Future studies are
needed to understand how Mesh1 functions to orchestrate these
diverse physiological responses in animals.
In conclusion, we have revealed the existence of the ppGpp hydrolase
Mesh1 in metazoa, along with its evolutionarily conserved structures
and biochemical functions. Moreover, we found that Drosophila Mesh1 is
involved in body growth and starvation responses, including gene expres-
sion regulation. We believe that our findings will open a new avenue in
understanding nutrient-dependent signaling pathways in metazoa.
Methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/nsmb/.
Accession codes. Protein Data Bank: Coordinates have been
­deposited with accession codes 3NR1 (human MESH1) and 3NQW
(Drosophila Mesh1).
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
Acknowledgments
This research was supported by grants from the Korean National Creative Research
Initiatives Program (2010-0018291) to J.C. and from the NMR Research Program
of Korea Basic Science Institute to Y.H.J. G.L. was supported by the Priority
Research Centers Program through the National Research Foundation of Korea,
funded by the Ministry of Education, Science and Technology (2008-005-J00203).
Y.K. and B.Y.K. were supported by World Class Institute Program of the National
Research Foundation of Korea, funded by the Ministry of Education, Science and
 articles
Technology. Wild-type (CF1648) and ΔrelA ΔspoT (CF1693) E. coli were kindly
provided by M. Cashel (US National Institutes of Health).
AUTHOR CONTRIBUTIONS
D.S. determined the structures and performed HPLC and MS analyses. G.L. and
J.H.L. performed Drosophila and bacteria experiments. H.-Y.K. and K.-J.K. helped
to determine the structures. H.-W.R. and J.-I.H. prepared PyDPA analyses. S.-Y.P.
analyzed nucleotides from larval extracts. Y.K. performed mammalian cell culture
experiments. All authors, including B.Y.K., C.P., H.E.C. and J.H.K. discussed the
experimental results and the text of the manuscript. D.S., G.L., J.H.L., Y.H.J. and
J.C. prepared the manuscript. J.C. and Y.H.J. developed the original research idea
and supervised the research project.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
1. Cashel, M. & Gallant, J. Two compounds implicated in the function of the RC gene
of Escherichia coli. Nature 221, 838–841 (1969).
2. Cashel, M., Gentry, D.R., Hernandez, V.J. & Vinella, D. The stringent response. in
Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology
© 2010 Nature America, Inc. All rights reserved.
1458–1496: (ASM Press, Washington, DC, 1996).
3. Potrykus, K. & Cashel, M. (p)ppGpp: still magical? Annu. Rev. Microbiol. 62,
35–51 (2008).
4. Haseltine, W.A. & Block, R. Synthesis of guanosine tetra- and pentaphosphate
requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the
acceptor site of ribosomes. Proc. Natl. Acad. Sci. USA 70, 1564–1568 (1973).
5. Battesti, A. & Bouveret, E. Acyl carrier protein/SpoT interaction, the switch linking
SpoT-dependent stress response to fatty acid metabolism. Mol. Microbiol. 62,
1048–1063 (2006).
6. Metzger, S. et al. Characterization of the relA1 mutation and a comparison of relA1
with new relA null alleles in Escherichia coli. J. Biol. Chem. 264, 21146–21152
(1989).
7. Xiao, H. et al. Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA
null mutants can be eliminated by spoT null mutations. J. Biol. Chem. 266,
5980–5990 (1991).
8. Laffler, T. & Gallant, J.A. Stringent control of protein synthesis in E. coli. Cell 3,
47–49 (1974).
9. Schreiber, G. et al. Overexpression of the relA gene in Escherichia coli. J. Biol.
Chem. 266, 3760–3767 (1991).
10. Kasai, K. et al. A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas
reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro.
Nucleic Acids Res. 30, 4985–4992 (2002).
11. Givens, R.M. et al. Inducible expression, enzymatic activity, and origin of higher
plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum.
J. Biol. Chem. 279, 7495–7504 (2004).
12. van der Biezen, E.A., Sun, J., Coleman, M.J., Bibb, M.J. & Jones, J.D. Arabidopsis
RelA/SpoT homologs implicate (p)ppGpp in plant signaling. Proc. Natl. Acad. Sci.
USA 97, 3747–3752 (2000).
13. Masuda, S. et al. The bacterial stringent response, conserved in chloroplasts,
controls plant fertilization. Plant Cell Physiol. 49, 135–141 (2008).
14. Mizusawa, K., Masuda, S. & Ohta, H. Expression profiling of four RelA/SpoT-like
proteins, homologues of bacterial stringent factors, in Arabidopsis thaliana. Planta
228, 553–562 (2008).
15. Tozawa, Y. et al. Calcium-activated (p)ppGpp synthetase in chloroplasts of land
plants. J. Biol. Chem. 282, 35536–35545 (2007).
16. Braeken, K. et al. New horizons for (p)ppGpp in bacterial and plant physiology.
Trends Microbiol. 14, 45–54 (2006).
17. Silverman, R.H. & Atherly, A.G. The search for guanosine tetraphosphate (ppGpp)
and other unusual nucleotides in eucaryotes. Microbiol. Rev. 43, 27–41 (1979).
18. Mittenhuber, G. Comparative genomics and evolution of genes encoding bacterial
(p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins). J. Mol.
Microbiol. Biotechnol. 3, 585–600 (2001).
19. Aravind, L. & Koonin, E.V. The HD domain defines a new superfamily of metal-
dependent phosphohydrolases. Trends Biochem. Sci. 23, 469–472 (1998).
20. Rhee, H.W. et al. Selective fluorescent chemosensor for the bacterial alarmone
(p)ppGpp. J. Am. Chem. Soc. 130, 784–785 (2008).
1194 VOLUME 17  NUMBER 10  OCTOBER 2010  nature structural & molecular biology
21. Gallant, J., Margason, G. & Finch, B. On the turnover of ppGpp in Escherichia coli.
J. Biol. Chem. 247, 6055–6058 (1972).
22. Takahashi, K., Kasai, K. & Ochi, K. Identification of the bacterial alarmone guanosine
5′-diphosphate 3′-diphosphate (ppGpp) in plants. Proc. Natl. Acad. Sci. USA 101,
4320–4324 (2004).
23. Guranowski, A. Metabolism of diadenosine tetraphosphate (Ap4A) and related
nucleotides in plants; review with historical and general perspective. Front. Biosci.
9, 1398–1411 (2004).
24. Holm, L. & Sander, C. Protein structure comparison by alignment of distance
matrices. J. Mol. Biol. 233, 123–138 (1993).
25. Zimmerman, M.D., Proudfoot, M., Yakunin, A. & Minor, W. Structural insight into
the mechanism of substrate specificity and catalytic activity of an HD-domain
phosphohydrolase: the 5′-deoxyribonucleotidase YfbR from Escherichia coli. J. Mol.
Biol. 378, 215–226 (2008).
26. Xu, R.X. et al. Atomic structure of PDE4: insights into phosphodiesterase mechanism
and specificity. Science 288, 1822–1825 (2000).
27. Mechold, U. et al. Functional analysis of a relA/spoT gene homolog from
Streptococcus equisimilis. J. Bacteriol. 178, 1401–1411 (1996).
28. Hogg, T. et al. Conformational antagonism between opposing active sites in a
bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the
stringent response. Cell 117, 57–68 (2004).
29. Avarbock, D., Avarbock, A. & Rubin, H. Differential regulation of opposing RelMtb
activities by the aminoacylation state of a tRNA.ribosome.mRNA.RelMtb complex.
Biochemistry 39, 11640–11648 (2000).
30. Wehmeier, L. et al. A Corynebacterium glutamicum mutant with a defined deletion
within the rplK gene is impaired in (p)ppGpp accumulation upon amino acid
starvation. Microbiology 147, 691–700 (2001).
31. Song, M. et al. ppGpp-dependent stationary phase induction of genes on Salmonella
pathogenicity island 1. J. Biol. Chem. 279, 34183–34190 (2004).
32. Artsimovitch, I. et al. Structural basis for transcription regulation by alarmone
ppGpp. Cell 117, 299–310 (2004).
33. Paul, B.J., Ross, W., Gaal, T. & Gourse, R.L. rRNA transcription in Escherichia coli.
Annu. Rev. Genet. 38, 749–770 (2004).
34. Magnusson, L.U., Farewell, A. & Nyström, T. ppGpp: a global regulator in Escherichia
coli. Trends Microbiol. 13, 236–242 (2005).
35. Durfee, T. et al. Transcription profiling of the stringent response in Escherichia coli.
J. Bacteriol. 190, 1084–1096 (2008).
36. Eymann, C., Homuth, G., Scharf, C. & Hecker, M. Bacillus subtilis functional
genomics: global characterization of the stringent response by proteome and
transcriptome analysis. J. Bacteriol. 184, 2500–2520 (2002).
37. Britton, J.S. & Edgar, B.A. Environmental control of the cell cycle in Drosophila:
nutrition activates mitotic and endoreplicative cells by distinct mechanisms.
Development 125, 2149–2158 (1998).
38. Beissbarth, T. & Speed, T.P. GOstat: find statistically overrepresented Gene
Ontologies within a group of genes. Bioinformatics 20, 1464–1465 (2004).
39. Palanker, L., Tennessen, J.M., Lam, G. & Thummel, C.S. Drosophila HNF4 regulates
lipid mobilization and beta-oxidation. Cell Metab. 9, 228–239 (2009).
40. Zinke, I. et al. Nutrient control of gene expression in Drosophila: microarray analysis
of starvation and sugar-dependent response. EMBO J. 21, 6162–6173 (2002).
41. Jones, G.H. & Bibb, M.J. Guanosine pentaphosphate synthetase from Streptomyces
antibioticus is also a polynucleotide phosphorylase. J. Bacteriol. 178, 4281–4288
(1996).
42. Hoyt, S. & Jones, G.H. relA is required for actinomycin production in Streptomyces
antibioticus. J. Bacteriol. 181, 3824–3829 (1999).
43. Mukai, J., Hirashima, A. & Mikuniya, T. Nucleotide 2′,3′-cyclic monophosphokinase
from actinomycetes. Nucleic Acids Symp. Ser. 22, 89–90 (1980).
44. Muta, S. et al. Streptomyces ATP nucleotide 3′-pyrophosphokinase and its gene.
Nucleic Acids Symp. Ser. 27, 165–166 (1992).
45. Sawaya, M.R. et al. Crystal structures of human DNA polymerase beta complexed
with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry
36, 11205–11215 (1997).
46. Ochi, K., Kandala, J.C. & Freese, E. Initiation of Bacillus subtilis sporulation by
the stringent response to partial amino acid deprivation. J. Biol. Chem. 256,
6866–6875 (1981).
47. Ochi, K. Streptomyces relC mutants with an altered ribosomal protein ST-L11 and
genetic analysis of a Streptomyces griseus relC mutant. J. Bacteriol. 172,
4008–4016 (1990).
48. Rhaese, H.J. Studies on the control of development synthesis of regulatory
nucleotides, HPN and MS, in mammalian cells in tissue cultures. FEBS Lett. 53,
113–118 (1975).
49. Thammana, P., Buerk, R.R. & Gordon, J. Absence of ppGpp production in
synchronised Balb/C mouse 3T3 cells on isoleucine starvation. FEBS Lett. 68,
187–190 (1976).
 ONLINE METHODS
Bacteria strains and plasmids. Wild-type (CF1648) and ΔrelA ΔspoT (CF1693)
E. coli were kindly provided by M. Cashel (US National Institutes of Health)7.
E. coli DH10β or BL21 (DE3) strains were used for protein production. For
bacterial transformation, the full-length relA gene was amplified from E. coli
(CF1648) genome and cloned into pGEX 4T-1 vector (Amersham). For in vitro
ppGpp synthesis, sequence encoding the 455 N-terminal amino acid residues
of RelA (s-relA)9 was cloned into pGEX 4T-1 vector. The full-length MESH1
(human HD domain containing-3, HDDC3; NCBI protein Q8N4P3.3) and
Mesh1 (Drosophila CG11900; NCBI protein NP_651682.1) cDNAs obtained
from Korean UniGene Information and the Drosophila Genomics Resource
Center, respectively, were cloned into the pGEX 4T-1 vector. For double plasmid
transformation27, we constructed chloramphenicol-resistant pG184 plasmid
by combining a PCR fragment from pGEX 4T-1 (954–3,058 bp) with a PCR
fragment from pACYC184 (1,577–3,567 bp, New England Biolabs). Then relA
and Mesh1 were cloned into pG184 vector containing Ptac promoter, glutathione
S-transferase (GST) tag sequence and p15A replication origin, and pGEX vector
containing Ptac, GST tag sequence and colE1 replication origin. For transfec-
tion into Drosophila S2 cells and HEK293T cells, Mesh1 and MESH1 were
respectively cloned into pRmHa-3 and pEBG vectors50 with hemagglutinin
(HA) tag sequences. To generate transgenic flies, relA and Mesh1 were cloned
into pUAST vector50 with HA tag sequence. For protein crystallization and
© 2010 Nature America, Inc. All rights reserved.
in vitro activity assays, MESH1 and Mesh1 were cloned into pET28a(+) vector
(Novagen). Sequence for RelSeq containing 385 N-terminal amino acids28 was
amplified from Streptococcus dysgalactiae subspecies equisimilis (obtained
from Korean Collection for Type Cultures) and cloned into pET28a(+)
vector. Point mutations were generated by QuikChange site-directed muta­
genesis kit (Stratagene). For protein expression and purification procedures,
see Supplementary Methods.
Crystallization, data collection and processing. Crystals of selenomethionyl
human MESH1 containing L43M and F147M mutations were obtained by hang-
ing drop vapor diffusion at 23 °C in a reservoir buffer containing 20%–25% PEG
3350 and 0.2 M sodium citrate. Structure of MESH1 was determined by SAD51,52.
The positions of selenium sites were located and refined by SOLVE53. Phases from
SAD phasing were further improved by solvent flattening using RESOLVE54. The
resultant electron density map was readily interpretable. Several rounds of iterative
model building using COOT55 and refinement using Refmac5 (ref. 56) and CNS57
were performed. Crystals of Drosophila Mesh1 were grown at 23 °C in a reservoir
buffer containing 100 mM sodium cacodylate, pH 6.5, and 1.2–1.5 M ammonium
sulfate. The structure of Mesh1 was solved by molecular replacement using the
structure of MESH1 as a search model with the program PHASER in the CCP4
program suite58. The model was completed by iterative cycles of model building
with COOT55 and refined with Refmac5 (ref. 56) and CNS57. The SAD and native
data sets were collected at 100K at beamlines 6C1 of Pohang Accelerator Laboratory
(Pohang, Korea). The Ramachandran plot generated by PROCHEK59 showed that
the model of MESH1 has 95.9% of residues in most favored regions and 4.1% of resi-
dues in additionally allowed regions; the model of Mesh1 has 90.3% of residues in
most favored regions and 8.8% of residues in additionally allowed regions. Table 1
lists data collection and refinement statistics of MESH1 and Mesh1.
Measurement of ppGpp hydrolysis by chemosensor PyDPA. The measurement
of ppGpp hydrolysis using PyDPA was performed as described20. A fluorescence
spectrophotometer (Varain Cary Eclipse) was used at 283 K with a 5-nm slit width
for excitation and emission. For the initial point, fluorescence emission spectra
(350 to 650 nm) were obtained upon excitation at 344 nm with 20 μM PyDPA
and 5 μM ppGpp (TriLink BioTechnologies) dissolved in 1 mM HEPES buffer,
pH 7.4. Mesh1 protein (0.5 μg) was added into 100 μl of PyDPA and ppGpp
solution, and the fluorescence emission spectra were monitored.
doi:10.1038/nsmb.1906
View publication stats
Measurement of ppGpp hydrolysis by thin-layer chromatography. To generate
radiolabeled ppGpp (3′-[β-32P]ppGpp)11, E. coli (DH10β) cells were transformed
with pGEX-s-relA (pGEX-relA for Supplementary Fig. 4b, right), treated with
1 mM IPTG at log phase for 1.5 h and lysed by sonication. The clarified lysates
containing GST-tagged RelA protein were incubated with GSH-agarose beads
(Peptron) at 4 °C for 1 h, and the beads were washed three times with PBS and
twice with buffer A (50 mM HEPES, pH 7.9, 250 mM NaCl, 2 mM EDTA, 14 mM
MgSO4, 1 mM β-mercaptoethanol). The beads were then incubated in 30 μl of
buffer A containing 10 mM GDP (Sigma-Aldrich) and 1 μCi of 5′-[γ-32P]ATP
(PerkinElmer, specific activity 6,000 Ci mmol−1) at 30 °C for 6 h. The supernatant
containing 3′-[β-32P]ppGpp was collected and stored at −20 °C for further use.
For the ppGpp hydrolysis assay, 1 μl of solution containing synthesized 3′-[β-32P]
ppGpp was incubated with Mesh1 protein (0.5 μg) in 30 μl of buffer B (50 mM
HEPES, pH 7.9, 250 mM NaCl, 14 mM MgSO4, 2.8 mM MnCl2, 1 mM β-mercapto­
ethanol) at 30 °C for 3 h (unless indicated otherwise). For Figure 2f (right gel),
Mn2+-free buffer B was used. After incubation, the reaction was stopped with one-
tenth volume of 88% (w/w) formic acid (Sigma-Aldrich). The samples were then
separated on polyethyleneimine TLC plates (Sigma-Aldrich) using 1.75 M sodium
phosphate as a mobile phase. Once the solvent front had reached 3 cm from the top,
the TLC sheets were air-dried and autoradiographed with a Bio-Imaging Analyzer
(FUJIX BAS IPR 2000). As standard markers, GDP, GTP (Sigma-Aldrich) and
ppGpp were loaded and separated on TLC, and then exposed to iodine.
Drosophila genetics. Mesh1 5A3 was generated by imprecise excision of a
P-element50 in the Mesh1 G3858 allele (GeniSys EP collection of BioMedical
Research Center, KAIST, Korea), whose genetic background is w1118. w1118 was
used as a wild-type control. For generating transgenic lines, pUAST-HA-relA and
pUAST-HA-Mesh1 plasmids were microinjected into w1118 embryos50. Other
stocks were obtained from the Bloomington Stock Center. See Supplementary
Methods for further experimental procedures.
Microscopy. Confocal images were acquired using a LSM 510 META laser scan-
ning microscope with LSM image browser version 3.2 SP2 software (Carl Zeiss).
Other microscopy images were acquired using a digital camera (AxioCam)
with AxioVS40AC version 4.4 software (Carl Zeiss). Images were processed in
Photoshop version 7.0 (Adobe).
50. Park, J. et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is
complemented by parkin. Nature 441, 1157–1161 (2006).
51. Gassner, N.C. & Matthews, B.W. Use of differentially substituted selenomethionine
proteins in X-ray structure determination. Acta Crystallogr. D Biol. Crystallogr. 55,
1967–1970 (1999).
52. Ohmura, T., Ueda, T., Hashimoto, Y. & Imoto, T. Tolerance of point substitution of
methionine for isoleucine in hen egg white lysozyme. Protein Eng. 14, 421–425
(2001).
53. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta
Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).
54. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol.
Crystallogr. 56, 965–972 (2000).
55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta
Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
56. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular
structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr.
53, 240–255 (1997).
57. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for
macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54,
905–921 (1998).
58. Collaborative Computational Project, Number 4. The CCP4 suite: programs for
protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763
(1994).
59. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a
program to check the stereochemical quality of protein structures. J. Appl. Cryst.
26, 283–291 (1993).
nature structural & molecular biology