Báo cáo khoa học: Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli pot

Characterization of the tRNA and ribosome-dependent
pppGpp-synthesis by recombinant stringent factor from
Escherichia coli
Rose-Marie Knutsson Jenvert
1,2
and Lovisa Holmberg Schiavone
1
1 Cell Biology Unit, Natural Science Section, So
¨
derto
¨
rns Ho
¨
gskola, Huddinge, Sweden
2 Department of Cell Biology, Arrhenius Laboratories E5, Stockholm University, Sweden
Prokaryotic cells coordinate the rate of mRNA, rRNA
and tRNA synthesis via the stringent response, which
is activated upon nutrient deprivation or stress
(reviewed in [1]). This physiological response is initi-
ated when stringent factor (SF) binds to translating
but stalled ribosomes that are starved for cognate
amino-acyl tRNAs. The stringent factor is activated by
the stalled ribosomal complex and starts to synthesize
the alarmone (p)ppGpp from GTP(GDP) using
ATP as a phosphate donor. Stringent factor is thus a
ribosome-dependent ATP:GTP pyrophosphoryl trans-
ferase that synthesizes (p)ppGpp. Production of this
alarmone results in a down-regulation of stable RNA
synthesis (rRNA and tRNA) and up-regulation of the
synthesis of mRNAs encoding enzymes involved in
amino acid biosynthesis.

The stringent factor was first isolated from ribo-
somal salt-wash fractions [2] and was identified as the
producer of magic spots I and II (ppGpp and
pppGpp [1]), in a (p)ppGpp synthesis assay. In this
assay, purified stringent factor is incubated with ribo-
somes, ATP and radiolabelled GTP. This is followed
by separation of newly synthesized pppGpp from
GTP by thin-layer chromatography [2]. The reported
Keywords
pppGpp; RelA; ribosome; stringent
response; tRNA
Correspondence
L. Holmberg Schiavone, Cell Biology Unit,
Natural Science Section, So
¨
derto
¨
rns
University College, S-141 89 Huddinge,
Sweden
Fax: +46 8608 4510
Tel: +46 8608 4597
E-mail:
(Received 25 August 2004, revised 4
November 2004, accepted 25 November
2004)
doi:10.1111/j.1742-4658.2004.04502.x
Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl trans-
ferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated
by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is

unclear how activation occurs. A His-tagged stringent factor was isolated
by affinity-chromatography and precipitation. This procedure yielded a
protein of high purity that displayed (a) a low endogenous pyrophosphoryl
transferase activity that was inhibited by the antibiotic tetracycline; (b) a
low ribosome-dependent activity that was inhibited by the A-site specific
antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a
tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp
per pmol stringent factor per minute. Footprinting analysis showed that
stringent factor interacted with ribosomes that contained tRNAs bound in
classical states. Maximal activity was seen when the ribosomal A-site was
presaturated with unacylated tRNA. Less tRNA was required to reach
maximal activity when stringent factor and unacylated tRNA were added
simultaneously to ribosomes, suggesting that stringent factor formed a
complex with tRNA in solution that had higher affinity for the ribosomal
A-site. However, tRNA-saturation curves, performed at two different ribo-
some ⁄ stringent factor ratios and filter-binding assays, did not support this
hypothesis.
Abbreviations
DMS, dimethylsulfate; SF, stringent factor; A-site, amino-acyl site; P-site, peptidyl-site; TC-ribosomes, twice salt-washed tight-couple
ribosomes; T4-mRNA, gene T4 mRNA-fragment.
FEBS Journal 272 (2005) 685–695 ª 2005 FEBS 685
activity of purified SF in the ribosome-dependent
reaction varies extensively, between 100 and 10 000
pmol pppGppÆpmol SF
)1
Æmin
)1
([3] and references
therein). The purified factor was also shown to dis-
play low activity in a ribosome-independent reaction

in the presence of 20% (v ⁄ v) alcohol [4].
Early on it was shown that unacylated tRNA in the
ribosomal amino-acyl site (A-site) stimulates pppGpp
synthesis by SF [5,6] and the general belief is that un-
acylated tRNA in the ribosomal A-site is required for
the activation of SF by ribosomes [5–7]. It is unclear
how unacylated tRNA enters the ribosomal A-site. At
least two options are possible: (a) it could enter the
A-site by simple diffusion; or (b) interaction of SF
with unacylated tRNA could increase the affinity of
the tRNA for the ribosomal A-site as originally sug-
gested by Richter [8].
It has also been shown that ribosomal protein L11 is
a stimulator of pppGpp synthesis in the ribosome-
dependent reaction [7,9,10]. Moreover, the A-site-speci-
fic antibiotic thiostrepton, which is dependent on L11
for ribosome-binding [11], inhibits ribosome-dependent
pppGpp synthesis in vitro [2]. Another A-site-specific
antibiotic, tetracycline, inhibits both ribosome-depend-
ent and independent pppGpp synthesis [6,12].
Altogether this shows that the function of SF is closely
connected to the ribosomal A-site, unacylated tRNA
and ribosomal protein L11 (see for example [7]) but it is
unclear how SF interacts with ribosomes and unacylated
tRNA and how pppGpp synthesis is stimulated.
We have isolated a recombinant His-tagged SF by
affinity-purification and by taking advantage of the
natural ability of the protein to form a precipitate [3].
The purified protein is highly active in a complete sys-
tem containing ribosomes, poly(U) and unacylated

tRNA
Phe
and converts approximately 4500 pmol GTP
to pppGppÆpmol SF
)1
Æmin
)1
. Here, the components
that are needed for pppGpp synthesis by SF are sys-
tematically mapped.
Results and Discussion
Stringent factor (SF) is a ribo some-dependent ATP:GTP
pyrophosphoryl transferase that is encoded by the relA
locus in Escherichia coli. We have cloned and purified
SF from E. coli and examined the ribosome, tem-
plate and tRNA-dependence of the pppGpp synthesis
reaction.
Purification of recombinant SF
We started out by purifying SF by affinity-chromato-
graphy using a His tag at the C-terminal end of the
protein and Ni-agarose beads according to Wendrich
et al. [7]. However, because SDS ⁄ PAGE analysis
showed that the resulting protein was contaminated by
low molecular mass proteins (Fig. 1, compare lanes 5–
7) SF was further purified by precipitation [3]. Several
low molecular mass contaminants were removed by
this procedure (Fig. 1, lanes 5–7), and the protein
could be further concentrated. The purified SF was
stored in the freezer, at a concentration of 1.0
mgÆmL

)1
in 20% (v ⁄ v) glycerol, without forming a pre-
cipitate or loss of activity. This is in contrast to the
results presented by Wendrich et al. [7] where purified
recombinant SF was found to precipitate at protein
concentrations exceeding 0.15 mgÆmL
)1
.
Activity of the recombinant protein
in the complete system
The activity of recombinant SF was first measured in
a complete system containing twice salt-washed tight-
couple ribosomes (TC-ribosomes), poly(U), tRNA
Phe
,
radiolabelled GTP and unlabelled substrates. In this
system (at 15 mm MgCl
2
) unacylated tRNA
Phe
may
bind to all of the tRNA binding sites on the ribosome
(i.e. A, P and E sites) [13,14]. As expected, the addi-
tion of SF to the system resulted in pppGpp synthesis
(Fig. 2). The speed of synthesis, calculated as des-
cribed in Experimental procedures, amounted to
4800 pmol pppGppÆpmol SF
)1
Æmin
)1

at the five-minute
Fig. 1. SDS ⁄ PAGE showing the purification of recombinant His-
tagged SF, indicated by the arrow. The cell extract containing over-
expressed SF (lane 1) was incubated with Ni-NTA agarose beads,
unbound protein was removed (lane 2) and the beads washed
(lanes 3, 4). SF was eluted with imidazole (lane 5) and precipitated
by dialysis against low salt ⁄ high magnesium buffer. The precipita-
ted protein was dissolved in high salt buffer and dialyzed into low
salt buffer (lane 7). Lane 6 shows protein contaminants that did not
precipitate and lane M contains protein markers. See Experimental
procedures for more details.
tRNA and ribosome-dependent synthesis by stringent factor R M. Knutsson Jenvert and L. Holmberg Schiavone
686 FEBS Journal 272 (2005) 685–695 ª 2005 FEBS
time-point. After that speeds dropped almost linearly
with time (Fig. 2, triangles). The drop in synthesis
speeds was caused by a shortage of substrate in the
reaction mixtures (Fig. 2, squares). It is possible that
the varying activity of SF reported in the literature
([3] and references therein) may in part be caused by
limiting supplies of nucleotides in the reaction mix-
tures, as the specific activity has often been measured
after long incubation times when nucleotides should
be limiting.
Endogenous activity of SF
The activity of SF in the presence of different transla-
tional components is summarized in Table 1. It is shown
that purified SF produced low amounts of pppGpp,
amounting to 150 pmol pppGppÆpmol SF
)1
Æmin

)1
,in
the presence of buffer and nucleotides. This synthesis is
visible in the autoradiogram in Fig. 3 (lane 14). That SF
has low synthetase ability in the absence of alcohol and
ribosomes is in accordance with an earlier report [3].
The endogenous activity of SF was not affected by the
addition of template or unacylated tRNA (Table 1) but
was inhibited by the antibiotic tetracycline (Fig. 3, lane
15). Similarly, the alcohol-activated assay is inhibited by
tetracycline [12]. The mechanism for this inhibition is
unknown [1].
Ribosome-dependence of SF in the complete
system
Table 1 shows that the addition of template, unacy-
lated tRNA and ribosomes to the activity assay stimu-
lated SF 30-fold when using optimal conditions. When
the ribosome-dependence was investigated more thor-
oughly it was found that SF activity increased with
increasing ribosome concentrations until a plateau was
reached at a 10-fold excess of ribosomes over SF
(results not shown). This is in accordance with results
presented by Wendrich et al. [7]. It is difficult to
understand the biological significance of the ribosome
Fig. 2. A time course of pppGpp synthesis by SF in the ribosome-
dependent reaction. Ribosomal complexes were formed by incuba-
ting TC-ribosomes (1.7 l
M) with poly(U) (0.16 lgÆlL
)1
) and tRNA

Phe
(10 lM) for 10 min at 37 °C. Radiolabelled GTP (0.6 lCi) was added
to the samples together with unlabeled substrates (10 m
M) and SF
(0.2 l
M). Samples were precipitated, with formic acid, at the indica-
ted times, and spotted on TLC plates to separate the nucleotides.
The pppGpp synthesis speeds (pmol pppGppÆpmol SF
)1
Æmin
)1
, m)
and available substrate concentrations (j) were calculated as des-
cribed in Experimental procedures and plotted as a function of
time. The input of radioactive GTP in the reactions is indicated by
the asterisk. See Experimental procedures for more details.
Table 1. Characterization of SF-activity in the poly(U)-dependent
system. Samples containing TC-ribosomes, poly(U) and tRNA
Phe
,
as indicated in the table, were incubated for 10 min at 37 °C
before addition of SF and nucleotides. Incubation was for 5 min
(complete system) or 20 min at 37 °C. Nucleotides were separ-
ated as described in the legend to Fig. 2 and the activity was
calculated as described in Experimental procedures. The values
are based on three independent experiments.
Ribosome
(1.7 l
M)
Poly(U)

(0.16 lgÆlL
)1
)
tRNA
Phe
(10 lM)
SF
(0.2 lM)
Activity (pmol
pppGppÆpmol
SF
)1
Æmin
)1
)
Activity
(% of
max)
+28±170
+163±333.5
+ + 169 ± 57 3.6
+ + 170 ± 72 3.6
+ + 332 ± 124 7.1
+ + + 327 ± 185 7.0
+ + + 342 ± 162 7.4
+ + + + 4461 ± 475 100
Fig. 3. A-site specific antibiotics inhibit the ribosome-dependent
activity in the absence of added tRNA. Autoradiograms showing the
inhibitory effects of viomycin, 0.1 m
M (lane 3), 1 mM (lane 4), 10 mM

(lane 5); tetracycline, (0.5 mM, lane 9); thiostrepton (10 lM, lane 10);
and micrococcin (10 l
M, lane 11) on TC-ribosome-dependent pppGpp
synthesis. Antibiotics were omitted from samples 2 and 8. Endo-
genous activity of SF (lanes 6, 14) in the presence of 10 m
M viomycin
(lane 7) and 0.5 m
M tetracycline (lane 15). Ribosomes were incuba-
ted with antibiotics before the addition of SF and nucleotides. Incuba-
tion was for 30 min at 37 °C. See Fig. 2 legend for more details.
R M. Knutsson Jenvert and L. Holmberg Schiavone tRNA and ribosome-dependent synthesis by stringent factor
FEBS Journal 272 (2005) 685–695 ª 2005 FEBS 687
titration curve as ribosomes should always be in molar
excess of SF [3,15,16] far exceeding the 10 : 1 ratio
that gives maximal synthesis speeds in the in vitro
assay (results not shown; [7]).
However, a model has been proposed to explain
how a few SF molecules can trigger the stringent
response within a few minutes on a large population
of ribosomes. In this model, SF molecules are sugges-
ted to hop between different stalled ribosomal com-
plexes and initiate pppGpp synthesis [7].
Ribosome-dependent but tRNA-independent
pppGpp synthesis?
Unacylated tRNA is incapable of stimulating SF in
the absence of ribosomes (Table 1) but do ribosomes
have an intrinsic ability of stimulating pppGpp synthe-
sis? This might have been overlooked in some earlier
studies where the activity of SF was 10-fold lower than
in the experiments presented here.

The stimulatory activity of ribosomes that had been
prepared by two different methods was analyzed: (a)
TC-ribosomes that are competent in binding 95%
tRNA in the ribosomal A-site [17]; and (b) ribosomes
that have been reassociated from subunits. The sub-
units have been exposed to low magnesium concentra-
tions during preparation [18] to dissociate ribosomes
with concomitant release of tRNA [5].
The activity of SF was found to increase threefold
in the presence of TC ribosomes (Fig. 4, lane 2;
450 pmol pppGppÆpmol SF
)1
Æmin
)1
) and twofold in
the presence of reassociated ribosomes (lane 3,
300 pmol pppGppÆpmol SF
)1
Æmin
)1
) compared to the
endogenous activity of the enzyme (lane 6). Moreover,
the TC-dependent activity was inhibited by antibiotics
that target pppGpp synthesis and ⁄ or the ribosomal
A-site (Fig. 3). Thus, viomycin (lanes 3–5), tetracycline
(lane 9), thiostrepton (lane 10) and micrococcin (lane
11) inhibited pppGpp synthesis compared to control
reactions carried out in the absence of antibiotics
(lanes 2 and 8).
Thiostrepton and micrococcin probably inhibit

pppGpp synthesis by blocking the function of L11
[7,9,19,20], whereas viomycin and tetracycline interfere
with A-site related functions [20–24]. As mentioned
earlier, tetracycline also inhibited SF in the absence of
ribosomes (Fig. 3, lanes 14–15; [1]), whereas the other
antibiotics did not inhibit this activity (Fig. 3, lanes
6–7; and results not shown).
It is known that stringent factor forms a stable com-
plex with ribosomes in the absence of unacylated
tRNA [7,8]. We speculate that formation of such com-
plexes stabilises SF and leads to the small production
of pppGpp visible in Figs 3 and 4 and that this ribo-
some-dependent activity is inhibited by antibiotics that
target the ribosomal A-site and ⁄ or protein L11.
However, it cannot be excluded that low levels
( 5%) of contaminating tRNAs in the ribosome pre-
paration caused the stimulatory effect. Here, it should
also be mentioned that in the complete system, reasso-
ciated ribosomes were 30% less efficient in stimulating
pppGpp synthesis than TC-ribosomes (Fig. 4, lanes
4–5). Similarly, reassociated ribosomes are not as com-
petent in binding tRNA [25] as TC-ribosomes [17].
Therefore, it appears that extensive purification of
ribosomes impairs the tRNA-binding and pppGpp
synthesis stimulating activity of ribosomes.
Template-dependence of pppGpp-synthesis
Table 1 shows that if SF is incubated with ribosomes,
unacylated tRNA and nucleotides, but no template,
the activity of the enzyme is similar to that in the
absence of tRNA. This is not surprising because the

template is needed to position tRNAs on the ribosome
[26].
The activity of the poly(U)-dependent system was
compared with a more natural system containing a
gene T4 mRNA fragment (from now on referred to as
Fig. 4. Ribosomes stimulate pppGpp synthesis by SF in the
absence of added tRNA. TC-ribosomes (1.7 l
M, lanes 2, 4) or 50S
(1.7 l
M) and 30S (2.3 lM) particles reassociated to 70S ribosomes
(lane 3, 5) were incubated with SF (0.3 l
M). Poly(U) (0.16 lgÆlL
)1
)
and tRNA (10 l
M) were added to samples 4 and 5 before the addi-
tion of SF. Endogenous activity of SF (lane 6). Incubation was for
10 min at 37 °C. See Fig. 2 legend for more details.
tRNA and ribosome-dependent synthesis by stringent factor R M. Knutsson Jenvert and L. Holmberg Schiavone
688 FEBS Journal 272 (2005) 685–695 ª 2005 FEBS
T4-mRNA [27]). The stimulatory activity of the
T4-mRNA programmed ribosomes was only 60% of
the poly(U)-programmed ribosomes in the presence of
added tRNA (Fig. 5, lanes 6–7). These results may be
explained by recent data where SF was found to inter-
act more strongly with poly(U) and full-length
mRNAs than with short mRNA fragments [7].
tRNA-dependence of pppGpp synthesis
Binding states of tRNAs
It was important to determine the binding states of

tRNAs in ribosomal complexes that stimulated
pppGpp synthesis by SF as tRNAs could either be
bound in classical or hybrid states [28]. First, it was
decided to monitor the state of the peptidyl site (P-site)
bound tRNA as this state determines the state of the
A-site bound tRNA.
Ribosomal complexes, containing tRNA
Met
f
, were
footprinted with dimethylsulfate (DMS) and kethoxal
at 15 mm MgCl
2
. Primer extension analysis of the
T4-mRNA programmed ribosomes showed that, at a
twofold excess of tRNA
Met
f
, there was a clear DMS
footprint at the E-site specific base C2394 in 23S
rRNA (Fig. 6A, red line) compared to samples con-
taining no tRNA (blue line). However, further analysis
revealed that this footprint disappeared when ribo-
somes were incubated with equimolar amounts of
unacylated tRNA
Met
f
(Fig. 6B, red line). Moreover,
chemical modification of tRNA
Met

f
-containing ribo-
somal complexes with kethoxal showed that strong
footprints were seen in the so called P-loop in 23S
rRNA at nucleotides G2252 and G2253 (Fig. 6C, red
line [28]). Altogether, this suggested that the ribosomal
complexes that interacted with SF in the activity assay
contained one tRNA
Met
f
that was bound in the P-site of
50S subunits and one tRNA
Met
f
that was bound in the
E-site of 50S subunits. The P-site bound tRNA
Met
f
also
gave clear DMS footprints on bases A794 and C795
in 16S rRNA that have been linked to the ribosomal
P-site of the 30S subunit (results not shown; [28]).
Addition of a threefold excess of tRNA
Phe
to the
tRNA
Met
f
programmed ribosomes resulted in a strong
Fig. 5. Autoradiograms showing the template and tRNA-depend-

ence of the pppGpp-synthesis reaction. The activity of TC-ribo-
somes (0.67 l
M, lane 2) containing T4-mRNA (1.3 lM, lane 3) plus
tRNA
Met
f
(1.3 lM, lane 4) plus tRNA
Phe
(6.7 lM, lane 5). Comparison
of activity of the poly(U) (2.45 lg, lane 6) and T4-mRNA (4 l
M, lane
7) dependent systems. Ribosomal complexes were formed by incu-
bating TC-ribosomes with T4-mRNA and tRNA
Met
f
for 10 min at
37 °C. tRNA
Phe
was added and incubation continued for 10 min. SF
was added to the reactions and samples were taken at 10 (lanes
2–5) and 5 (lanes 6, 7) min. See Fig. 2 legend for more details.
A
B
C
D
E
Fig. 6. Footprinting analysis of the interaction of tRNA
Met
f
(A, B, C)

and tRNA
Phe
(D, E) with the T4-mRNA programmed TC-ribosomes.
Complexes were formed with twofold excess (A, C, D, E) or equi-
molar amounts of tRNA
Met
f
(B) before addition of threefold excess
of tRNA
Phe
(D, E). DMS (A, B, E) and kethoxal (C, D) modifications
and primer extensions were performed according to Experimental
procedures. Mock-modified control (black line), samples without
added tRNA (blue lines) and samples containing tRNAs (red lines).
See Fig. 5 legend for more details.
R M. Knutsson Jenvert and L. Holmberg Schiavone tRNA and ribosome-dependent synthesis by stringent factor
FEBS Journal 272 (2005) 685–695 ª 2005 FEBS 689
A-site footprint at G530 in 16S rRNA (Fig. 6D, red
line [28]) and A-site footprints in domain IV in 23S
rRNA at nucleotides C1941 and C1942 (Fig. 6E, red
line [29]). A footprint was also seen at position A1966
by binding of tRNA
Met
f
to the P-site of 50S subunits
(visible in Fig. 6E).
From these results it can be concluded that tRNAs
were bound in classical states with the tRNA
Met
f

bound
in the P-sites of the 30S and 50S subunits and the
tRNA
Phe
bound in the A-site of the 30S and 50S sub-
unit [28]. Moreover, a tRNA
Met
f
was present in the 50S
E-site. The E-site bound tRNA did not affect the stim-
ulatory activity of ribosomes as ribosomal complexes
formed with 1.2-fold or twofold excess of tRNA
Met
f
sti-
mulated SF to the same extent upon addition of
tRNA
Phe
to the activity assay (results not shown).
Binding of tRNA
Met
f
to ribosomes
Binding of tRNA
Met
f
to T4-mRNA programmed ribo-
somes did not increase the ability of ribosomes to sti-
mulate pppGpp-synthesis (Fig. 5, lane 4). This is in
agreement with other data [5,7] and thus supports the

notion that unacylated tRNA has to be bound in the
ribosomal A-site for activation of SF to occur.
Titration of tRNA
phe
to the A-site of T4-mRNA
programmed ribosomes
T4-mRNA-programmed TC-ribosomes, containing
tRNA
Met
f
, were incubated with increasing amounts of
tRNA
Phe
before the addition of SF. As can be seen in
Fig. 7A, the activity of SF, calculated as pmol
pppGppÆpmol ribosome
)1
Æmin
)1
, increased with
increasing amounts of tRNA
Phe
added until a plateau
was seen at a threefold molar excess of tRNA
Phe
over
ribosomes (Fig. 7A). This shows that the ribosomal
A-site was saturated with unacylated tRNA [14] when
maximal activation of SF occurred.
Binding of tRNA

Phe
to the A, P and E-sites of poly(U)
programmed ribosomes
In the second set of experiments poly(U) programmed
ribosomes were incubated with increasing amounts of
tRNA
Phe
before addition of SF. In Fig. 7B it can be
seen that the tRNA-binding curves reached a plateau
at a fivefold to 10-fold molar excess of tRNA
Phe
over
ribosomes. Thus, in this system, more tRNA
Phe
was
required to get maximal SF activity. This is not surpri-
sing because tRNA
Phe
will bind to all three tRNA bind-
ing sites on poly(U) programmed ribosomes [14] and
two molar equivalents of tRNA are needed to saturate
Fig. 7. tRNA-titration curves showing the tRNA-dependence of the
pppGpp synthesis reaction. pppGpp synthesis speeds (pmol
pppGppÆpmol ribosome
)1
Æmin
)1
) were plotted as a function of dif-
ferent tRNA ⁄ ribosome ratios ⁄ TC-ribosomes (0.67 l
M) programmed

with (A) T4-mRNA + tRNA
Met
f
or (B, C) poly(U). (A, B) tRNA
Phe
was
added, at the indicated concentrations, and incubation continued
for 10 min at 37 °C. SF was added at two different concentrations
(0.13 l
M, ; or 0.67 lM, m) together with tRNA
Phe
(C) and the incu-
bation continued for 5 (A) or 10 (B, C) min at 37 °C. Nucleotides
were separated and speeds (pmol pppGppÆpmol ribosome
)1
Æmin
)1
)
calculated as described in Experimental procedures. The curves are
based on at least three independent experiments. Refer to the leg-
ends of Figs 2 and 6 for more details.
tRNA and ribosome-dependent synthesis by stringent factor R M. Knutsson Jenvert and L. Holmberg Schiavone
690 FEBS Journal 272 (2005) 685–695 ª 2005 FEBS
the P- and E-sites before A-site binding [13]. It can also
be concluded from this experiment that maximal
SF-activation required that the A-site was saturated
with unacylated tRNA.
Is the tRNA-stimulatory effect affected by the order
of addition of tRNA and SF to the reactions?
The two types of tRNA titration experiments showed

that ribosomes must be saturated with tRNA
Phe
for
maximal activation of SF to occur. A-site bound
unacylated tRNA
Phe
should be stably bound in the
experiments presented here as the half-life of dissoci-
ation is more than 2 h [14]. The strong footprint at
G530 in 16S rRNA supports this notion (Fig. 6E).
This can be compared to the weak A-site binding
needed for maximal SF activation in the system used
by Wendrich et al. [7]. Curiously, there was one dif-
ference it the way that the experiments were per-
formed, as in that system recombinant SF was added
together with unacylated tRNA
Phe
to ribosomes,
whereas in our system ribosomes were preincubated
with unacylated tRNA
Phe
before the addition of SF.
Is it possible that less tRNA is needed to reach
maximal activation of SF by adding SF and tRNA
together to ribosomes?
To investigate this, it was tested whether the tRNA-
saturation curve would behave differently by adding
SF and tRNA
Phe
simultaneously to poly(U)-pro-

grammed ribosomes. Surprisingly, the experiments sug-
gest that this prediction is true. The curves in Fig. 7C
show that saturation was reached at a two- to threefold
molar excess of tRNA
Phe
over ribosomes when SF was
added together with tRNA
Phe
to the activity assay. In
this last experiment the ribosomal A-site would not be
saturated with tRNA
Phe
as P- and E-site binding pre-
cedes A-site binding [13] and two molar equivalents of
tRNA
Phe
are needed to saturate the ribosomal P- and
E-sites. The experiments suggest therefore that only
weak binding of unacylated tRNA
Phe
in the ribosomal
A-site was needed when SF and tRNA were added sim-
ultaneously to the reaction mixtures, in agreement with
the results presented by Wendrich et al. [7].
The stimulatory effect of tRNA was not affected
by two different ribosome ⁄ SF ratios tested
We found it intriguing that the order of addition of
tRNA and SF to the activity assay affected the tRNA-
saturation curve. This suggested that SF might form a
complex with unacylated tRNA in solution that has

higher affinity for ribosomes than unacylated tRNA
by itself, as originally suggested by Richter [8]. If so,
the tRNA saturation curves might be affected by the
amount of SF present in the reaction.
Therefore, the tRNA-saturation curves were per-
formed at two different ribosome ⁄ SF ratios: first, a
fivefold molar excess of ribosomes (Fig. 7, triangles);
or second, equimolar amounts of ribosomes and SF
(Fig. 7, rectangles). (The concentration of ribosomes
was constant in the experiments whereas the SF con-
centration varied.)
The results show that maximal activity of SF was
reached at similar tRNA levels independent of the ribo-
some ⁄ SF level (Fig. 7). This was true for both the T4-
mRNA-dependent system and the poly(U)-dependent
systems. Therefore, this experiment does not support
the notion that SF should form a complex with tRNA
in solution before binding to ribosomes because similar
amounts of tRNA were needed independent of the SF
concentration. The higher activity of the systems con-
taining more SF (1 : 1 ratio between SF and ribosome)
may be attributed to the endogenous activity of SF
(150 pmol pppGppÆpmol ribosome
)1
Æmin
)1
).
Does SF form a complex with unacylated tRNA
in solution?
We also tried to isolate a complex between tRNA and

SF in solution by filter-binding assays. In this assay,
SF was incubated with tritium-labelled unacylated
tRNA in a buffer containing 20 mm MgCl
2
and vary-
ing salt concentrations (Table 2). Reactions were
chilled on ice before filtration through a 0.45 lm Milli-
pore filter. The results show that approximately 10-fold
more tRNA was retained on the filter in the presence
of SF (Table 2). The effect was specific to SF, as incu-
bation of unacylated tRNA with recombinant ribo-
somal protein L10 or ovalbumin did not increase the
amount of tRNA retained on the filter (results not
shown). Binding was slightly more efficient at lower
(50 mm) than higher (100 mm) salt concentrations.
Table 2. Binding of unacylated tRNA to SF in solution. SF-tRNA
complexes were formed as described in Experimental procedures
and complexes were separated from unbound tRNA by filter-
binding. The values are dependent on at least three independent
experiments.
Salt
[KCl]
tRNA
(pmol)
SF
(pmol)
Binding
(nCi)
tRNA bound
(%)

100 60 – 777 ± 247 0.3
100 30 20 4019 ± 653 3.4
100 60 20 3849 ± 566 1.6
50 60 – 756 ± 127 0.3
50 30 20 5736 ± 770 4.8
50 60 20 5774 ± 628 2.4
R M. Knutsson Jenvert and L. Holmberg Schiavone tRNA and ribosome-dependent synthesis by stringent factor
FEBS Journal 272 (2005) 685–695 ª 2005 FEBS 691
However, the amount of tRNA retained was low
compared to amounts of SF and total tRNA present in
the reaction (Table 2). Moreover, similar levels of
tRNA were bound at the two concentrations of tRNA
tested. It is difficult to estimate the significance of the
data because it cannot be ruled out that tRNA was
trapped on filters through a nonspecific interaction with
SF. Of course, it is possible that SF formed a labile
complex with tRNA in solution and that this complex
was prone to dissociate upon dilution of the reactions
before filtration, in analogy with the weak interaction
of unacylated tRNA with the ribosomal E-site [30]. We
are currently investigating this theory more thoroughly.
Levels of unacylated tRNA are increased during the
stringent response (reviewed in [1]). Experimental data
suggest that unacylated tRNA interacts with the 30S
A-site in vivo [31] but it is not understood how the
tRNA is directed to the A-site in the cell. The data
presented here does not support the hypothesis that
SF directs unacylated tRNA to the ribosomal A-site
for the following reasons: the tRNA-saturation curves
were independent of the two ribosome ⁄ SF ratios tes-

ted, and we were unable to isolate significant amounts
of a putative tRNA–SF complex by filter-binding assays.
In vitro, it is easy to manipulate binding of unacylated
tRNA to the ribosome by increasing the magnesium
concentration [14]. The magnesium concentration also
affects the binding states of the tRNAs on the ribosome.
Unacylated tRNAs can be bound in either classical or
hybrid states [28]. We have shown here by footprinting
analysis that in our buffer system at 15 mm MgCl
2
SF
interacted with ribosomes that contain tRNAs bound in
classical states. This means that the 50S A- and P-sites
interacted with the 3¢ end of unacylated tRNA
Phe
and
tRNA
Met
f
, respectively. Thus, in this study, SF was acti-
vated by unacylated tRNA
Phe
that sits in the 50S A-site.
In contrast, if the tRNAs had been bound in hybrid
states the CCA end of unacylated tRNA
Phe
would have
been bound in the 50S P-site and the 50S A-site would
have been empty [28].
Most SF activity studies have been performed at 10–

20 mm Mg
2+
[2,6,8,15,32,33] although Wendrich et al.
[7] performed their studies at 6 mm Mg
2+
with addi-
tional spermine and spermidine. It is therefore imposs-
ible to say whether tRNAs were bound in similar states
in all of the above studies. In the cell, SF probably
binds to a ribosome with a peptide in the P-site (P ⁄ P-
state) and an unacylated tRNA in the A-site. This unac-
ylated tRNA must therefore bind in the A ⁄ A-state (in
analogy with this study) although, to our knowledge,
the interaction of unacylated tRNA with ribosomes that
are filled with peptide has not been structurally mapped.
Haseltine and Block [5] used this type of ribosomal
complex when they discovered the stimulatory effect of
adding unacylated tRNA to the ribosomal A-site. It
would be interesting to compare the kinetics of SF in
the physiological system with the system used here, con-
taining only unacylated tRNAs. Here, it should also be
mentioned that it has been suggested that the 50S sub-
unit may contain a domain that senses the aminoacyla-
tion state of the tRNA in analogy with the T-box in
antitermination of transcription of amino acid biosyn-
thetic enzymes [1,34]. We suggest that a putative T-box
on the 50S subunit would be part of the 50S A-site, as
unacylated tRNA is required for stimulation of SF by
ribosomes and this tRNA sits in the 50S A-site.
The ribosome-dependence of SF has been known

since the factor was first isolated more than 30 years
ago. Despite this fact, there are still big gaps in our
knowledge of how SF interacts with the ribosome and
which ribosomal components are essential for the acti-
vation of pppGpp synthesis. This might be partly due to
the fact that SF is present in very low amounts in the
cell [3,15] and has therefore been hard to purify. During
the last few years, several different recombinant pyro-
phosphoryl transferase have been cloned and isolated
([7,35]; this study). Work with the recombinant proteins
have elucidated the endogenous activity of SF ([35]; this
study), and how SF interacts with ribosomes ([7,35]; this
study). Future experiments will reveal how SF binds to
and is activated by ribosomes and unacylated tRNA.
Experimental procedures
Materials
tRNA
Met
f
was from Boehringer (Ingelheim, Germany) and
tRNA
Phe
, poly(U), ATP, GTP, isopropyl thio-b-d-galacto-
side and polyethyleneimine plates (Macherey & Nagel,
Du
¨
ren, Germany) were from Sigma-Aldrich (St. Louis,
MO, USA).
3
H-labelled acylated tRNA was prepared and

stripped of amino acid according to [36]. The phage T4
gene 32 mRNA fragment was from Dharmacon (Lafayette,
CO, USA). The sequence of the fragment is according to
[27]. [
32
P]dGTP[aP] (10 mCiÆmL
)1
) and Hyperfilm MP was
from Amersham Bioscience (Buckinghamshire, UK). DMS
was from Sigma, kethoxal was from ICN (Irvine, CA,
USA), Superscript reverse transcriptase was from Life
Technologies, Inc. (Rockville, MD, USA) and the DNA
sequencing kit was from PerkinElmer (Boston, MA, USA).
Cloning of the E. coli relA gene
The relA gene was amplified by PCR from E. coli MRE 600
genomic DNA with primers 5¢-CGGGAATTCCATATGGT
TGCGGTAAGAAT-3¢ and 5¢-CCCGCTCGAGACTCCCG
tRNA and ribosome-dependent synthesis by stringent factor R M. Knutsson Jenvert and L. Holmberg Schiavone
692 FEBS Journal 272 (2005) 685–695 ª 2005 FEBS
TGCAACCGACG-3¢ containing NdeI and XhoI recognition
sequences, respectively, and inserted into a TOPO-vector
(Invitrogen, Carlsbad, CA, USA). Afterwards, the gene was
subcloned into the pET24b vector to generate pET24b(relA).
The correct sequence of relA was confirmed by sequencing
using the primers 5¢-AGCAATACGCTCCGCCAG-3¢,
5¢-TGGCGGATGCCAACGTAG-3¢,5¢-CTCGACCGCGA
ACACTAC-3¢,5¢-CACCCAACTCTGCATCTTC-3¢,5¢-TT
TCGAACGCCCACGGC-3¢ and 5¢-TGTACTGAAATACC
GCGCC-3¢.
Expression and purification of stringent factor

SF was purified from BL21(DE3) cells, grown in 2· YT
medium, containing the pET24b(relA) plasmid. Protein
expression was induced with 0.5 mm isopropyl thio-b-d-
galactoside, at D
550
¼ 0.7, for 4 h at 30 °C. Cells were har-
vested by centrifugation (11 000 g, 16 min, 4 °C) and
washed with 0.1 m NaCl, 10 mm Tris ⁄ HCl pH 8.0, 1 mm
EDTA. The cell pellet was dissolved in lysis buffer: 50 mm
NaH
2
PO
4
, 300 mm NaCl, 10 mm imidazole, 10% (v ⁄ v) gly-
cerol, 10 mm 2-mercaptoethanol, pH 8.0. Lysosyme
(1 mgÆmL
)1
) was added and the cell suspension was left on
ice for 30 min before sonication (10 · 15 s, 2 · 30 s, 1 min
between cycles). Cell debris was removed by two centrifuga-
tions for 15 min at 23 000 g,4°C. The cleared cell lysate
was incubated with Ni-NTA agarose beads (Qiagen,
Valencia, CA, USA) for 1 h at 4 °C and washed four times
with lysis buffer containing 1 m NaCl and 20 mm imidazole.
The suspension was transferred to a column and SF was elut-
ed in 0.5 mL fractions with lysis buffer containing 250 mm
imidazole. Fractions containing protein were dialyzed over-
night against 10 mm Tris ⁄ HCl pH 8.0, 14 mm MgOAc,
60 mm KOAc, 0.5 mm EDTA, 10% (v ⁄ v) glycerol, 10 mm
2-mercaptoethanol. Using these conditions SF formed a pre-

cipitate [3]. The precipitate was dissolved in 10 mm Tris ⁄ HCl
pH 8.0, 1 m KCl, 1 mm EDTA, 10% (v ⁄ v) glycerol, 10 mm
2-mercaptoethanol and dialyzed overnight against SF buffer:
30 mm Hepes pH 8.0, 300 mm KCl, 20% (v ⁄ v) glycerol and
10 mm 2-mercaptoethanol. The protein concentration was
determined according to Bradford and aliquots of the protein
were quick-frozen and stored at )80 °C. The His tag did not
appear to interfere with the activity of the protein, as the
recombinant SF was highly active in accordance with previ-
ous results [7].
Purification of ribosomes
Tight-couple ribosomes from E. coli strain MRE600 were
purified according to [17], except that cells were lysed by
sonication (6 · 15 s; 2 · 20 s, 1 min between cycles). Ribo-
somes were suspended in 20 mm Tris ⁄ HCl (pH 7.6), 10 mm
MgCl
2
, 100 mm NH
4
Cl, 0.5 mm EDTA, 6 m m 2-mercapto-
ethanol and stored in small aliquots at )80 °C. Ribosomal
30S and 50S subunits were purified according to [37]. The
purity of ribosomes was checked by denaturing gels con-
taining 8 m urea and the activity of ribosomes was tested in
poly(Phe) synthesis assays according to [38].
pppGpp synthesis
pppGpp synthesis assays were essentially carried out
according to Haseltine and Block [5] with the following
modifications. In the standard assay TC-ribosomes
(25 pmol) were programmed with poly(U) (2.45 lg) and

tRNA
Phe
(150 pmol) in a buffer containing 20 mm MgCl
2
,
100 mm KCl, 30 mm Hepes pH 8.0, 10 mm 2-mercaptoeth-
anol for 10 min at 37 °C. A mixture of ATP and GTP
(10 mm final concentrations) and [
32
P]GTP[aP] (0.6 lCi)
was added to the reactions and the MgCl
2
concentration
was adjusted to 15 mm. SF (5 pmol, unless otherwise indi-
cated) was added to the reaction mixtures (total volume
15 lL) and incubation was continued for the indicated
times at 37 °C. T4-mRNA, tRNA
Met
f
and tRNA
Phe
were
used at the concentrations indicated in the figures. In some
assays TC-ribosomes were preincubated with the antibiotics
tetracycline (500 lm), micrococcin (10 lm), thiostrepton
(10 lm) and viomycin (0.1–10 mm) before addition of
nucleotides and SF. The reactions were stopped by the
addition of 1 lL 88% (v ⁄ v) formic acid, incubated on ice
for 15 min and centrifuged for 5 min at 16 000 g in an epp-
endorf centrifuge at 4 °C.

Separation of pppGpp from GTP and calculation
of synthesis speeds
Radiolabelled nucleotides were separated by thin layer
chromatography. Supernatants (10 lL) were spotted on
polyethyleneimine cellulose plates and the nucleotides were
allowed to migrate using 1.5 m KH
2
PO
4
(pH 3.4) as a buf-
fer. The radioactive spots corresponding to GTP and
pppGpp were identified by autoradiography using a phos-
phorimager or Hyperfilm MP. The amounts of pppGpp
synthesized were quantified by phosphorimager analysis or
by counting radioactivity in a liquid scintillation counter
after the spots were cut out. Turnover rates were calculated
as percent of radioactive (p)ppGpp of the total amount of
radioactivity. This was then normalized in relation to the
time and amount of SF ⁄ ribosome used, yielding the rate
pmol pppGppÆ pmol SF
)1
Æmin
)1
or pmol pppGppÆpmol
ribosome
)1
Æmin
)1
.
Chemical modification and primer extension

analysis
TC-ribosomal complexes containing T4-mRNA, tRNA
Met
f
and tRNA
Phe
were modified with DMS according to [39].
Alternatively, samples were modified with kethoxal (18 mm
final concentration) for 15 min at 37 °C. The kethoxal
R M. Knutsson Jenvert and L. Holmberg Schiavone tRNA and ribosome-dependent synthesis by stringent factor
FEBS Journal 272 (2005) 685–695 ª 2005 FEBS 693
adduct was stabilized with 25 mm K-borate (pH 7.0) at all
times. RNA was precipitated with ethanol and extracted
from protein according to [39]. The positions of the modi-
fied sites were identified by primer extension according to
[40]. The primers used were according to [28] except that a
fluorescent label was included at the 5¢ end of the probe.
The following primers were used: 5 ¢-CCGAACTGTCT
CACGAC-3¢ (906, 16S rRNA), 5¢-TGTTATCCCCGGAG
TAC-3¢ (2437, 23S rRNA), 5¢-GCATTTCAC CGCT ACAC -3¢
(683, 16S rRNA) and 5¢-TCCGTCTTGCCGCGGGT-3¢
(2042, 23S rRNA). The primer extension products were
analyzed on 5% (w ⁄ v) polyacrylamide sequencing gels in
an Applied Biosystems 377 DNA sequencer as described
previously [40].
Filter-binding assays
SF (20 pmol) was incubated with
3
H-labelled unacylated
tRNA (30 or 60 pmol; specific activity 1.7 nCiÆpmol

)1
)ina
buffer containing 50 mm KCl, 20 mm MgOAc, 30 mm He-
pes pH 7.8, 0.5 mm EDTA and 6 mm 2-mercaptoethanol
for 10 min at 30 °C. The final volume of the reactions was
10 lL. Reactions were cooled on ice for 10 min, diluted to
1.5 mL with the same buffer and filtered through a
0.45 lm Millipore filter. Filters were washed with
3 · 1.5 mL buffer and the samples were counted in a liquid
scintillation counter.
Acknowledgements
Odd Nyga
˚
rd is thanked for critical reading of the
manuscript and general support. Ma
˚
ns Ehrenberg is
thanked for helpful discussions and Harry Noller is
thanked for insightful comments. This research is sup-
ported by a grant from the Swedish Research Council
(Dnr 5 ⁄ 42 ⁄ 2001).
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