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-
Trang 1Characterization of the tRNA and ribosome-dependent
pppGpp-synthesis by recombinant stringent factor from Escherichia coli
Rose-Marie Knutsson Jenvert1,2and Lovisa Holmberg Schiavone1
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: lovisa.holmberg-schiavone@sh.se
(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.
Trang 2activity of purified SF in the ribosome-dependent
reaction varies extensively, between 100 and 10 000
pmol pppGppÆpmol SF)1Æmin)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
tRNAPhe and converts approximately 4500 pmol GTP
to pppGppÆpmol SF)1Æmin)1 Here, the components
that are needed for pppGpp synthesis by SF are
sys-tematically mapped
Results and Discussion
Stringent factor (SF) is a ribosome-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)1in 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), tRNAPhe, radiolabelled GTP and unlabelled substrates In this system (at 15 mm MgCl2) unacylated tRNAPhe 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)1Æmin)1at 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.
Trang 3time-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)1Æmin)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 l M ) 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 spotindica-ted on TLC plates to separate the nucleotides.
The pppGpp synthesis speeds (pmol pppGppÆpmol SF)1Æmin)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 l M ) SF (0.2 l M )
Activity (pmol pppGppÆpmol
SF)1Æmin)1)
Activity (% of max)
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 m M (lane 4), 10 m M
(lane 5); tetracycline, (0.5 m M , lane 9); thiostrepton (10 l M , 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.
Trang 4titration 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)1Æmin)1) and twofold in
the presence of reassociated ribosomes (lane 3,
300 pmol pppGppÆpmol SF)1Æmin)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 l M ) 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) Incubaaddi-tion was for
10 min at 37 C See Fig 2 legend for more details.
Trang 5T4-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 tRNAMetf , were
footprinted with dimethylsulfate (DMS) and kethoxal
at 15 mm MgCl2 Primer extension analysis of the
T4-mRNA programmed ribosomes showed that, at a
twofold excess of tRNAMet
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 tRNAMetf (Fig 6B, red line) Moreover, chemical modification of tRNAMetf -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 tRNAMet
f that was bound in the P-site of 50S subunits and one tRNAMet
f that was bound in the E-site of 50S subunits The P-site bound tRNAMet
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 tRNAPhe to the tRNAMet
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 l M , lane 3) plus
tRNA Met
f (1.3 l M , lane 4) plus tRNA Phe (6.7 l M , 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.
Trang 6A-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 tRNAMet
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 tRNAMetf bound
in the P-sites of the 30S and 50S subunits and the
tRNAPhe bound in the A-site of the 30S and 50S
sub-unit [28] Moreover, a tRNAMetf 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 tRNAMet
f sti-mulated SF to the same extent upon addition of
tRNAPheto the activity assay (results not shown)
Binding of tRNAMetf to ribosomes
Binding of tRNAMet
f to T4-mRNA programmed ribo-somes did not increase the ability of riboribo-somes 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 tRNApheto the A-site of T4-mRNA
programmed ribosomes
T4-mRNA-programmed TC-ribosomes, containing
tRNAMet
f , were incubated with increasing amounts of
tRNAPhe before the addition of SF As can be seen in
Fig 7A, the activity of SF, calculated as pmol
pppGppÆpmol ribosome)1Æmin)1, increased with
increasing amounts of tRNAPhe added until a plateau
was seen at a threefold molar excess of tRNAPhe 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 tRNAPheto 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
tRNAPhe 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 tRNAPhe over
ribosomes Thus, in this system, more tRNAPhe was
required to get maximal SF activity This is not
surpri-sing because tRNAPhewill 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)1Æmin)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 l M , 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)1Æmin)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.
Trang 7the 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 tRNAPhe for
maximal activation of SF to occur A-site bound
unacylated tRNAPhe 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 tRNAPhe to ribosomes,
whereas in our system ribosomes were preincubated
with unacylated tRNAPhe 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 tRNAPhe 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 tRNAPheover ribosomes when SF was
added together with tRNAPhe to the activity assay In
this last experiment the ribosomal A-site would not be
saturated with tRNAPhe as P- and E-site binding
pre-cedes A-site binding [13] and two molar equivalents of
tRNAPhe are needed to saturate the ribosomal P- and
E-sites The experiments suggest therefore that only
weak binding of unacylated tRNAPhein 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)1Æmin)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 MgCl2 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 (%)
Trang 8However, 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 MgCl2SF
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 tRNAPhe and
tRNAMet
f , respectively Thus, in this study, SF was
acti-vated by unacylated tRNAPhethat sits in the 50S A-site
In contrast, if the tRNAs had been bound in hybrid
states the CCA end of unacylated tRNAPhewould 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 Mg2+[2,6,8,15,32,33] although Wendrich et al
[7] performed their studies at 6 mm Mg2+ 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 tRNAMet
f was from Boehringer (Ingelheim, Germany) and tRNAPhe, 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) 3H-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] [32P]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
Trang 9TGCAACCGACG-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 D550¼ 0.7, for 4 h at 30 C Cells were
har-vested by centrifugation (11 000 g, 16 min, 4C) 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
NaH2PO4, 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, 4C The cleared cell lysate
was incubated with Ni-NTA agarose beads (Qiagen,
Valencia, CA, USA) for 1 h at 4C 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 prepre-cipitate 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
MgCl2, 100 mm NH4Cl, 0.5 mm EDTA, 6 mm
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 tRNAPhe(150 pmol) in a buffer containing 20 mm MgCl2,
100 mm KCl, 30 mm Hepes pH 8.0, 10 mm 2-mercaptoeth-anol for 10 min at 37C A mixture of ATP and GTP (10 mm final concentrations) and [32P]GTP[aP] (0.6 lCi) was added to the reactions and the MgCl2 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 37C T4-mRNA, tRNAMet
f and tRNAPhe 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 4C
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 KH2PO4(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 pppGppÆpmol SF)1Æmin)1 or pmol pppGppÆpmol ribosome)1Æmin)1
Chemical modification and primer extension analysis
TC-ribosomal complexes containing T4-mRNA, tRNAMet
f and tRNAPhewere modified with DMS according to [39] Alternatively, samples were modified with kethoxal (18 mm final concentration) for 15 min at 37C The kethoxal
Trang 10adduct 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 identimodi-fied 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¢-GCATTTCACCGCTACAC-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 3H-labelled unacylated
tRNA (30 or 60 pmol; specific activity 1.7 nCiÆpmol)1) in a
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 30C 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)
References
1 Cashel M, Gentry DR, Hernandez VJ & Vinella D
(1996) The stringent response In Esherichia coli and
Salmonella: Cellular and Molecular Biology
(Neider-hardt FC, Curtis R, Ingraham JL, Lin ECC, Low
KB, Magasanik B, Reznikoff WS, Riley M,
Schaech-ter M & Umbarger HE, eds), pp 1458–1496 ASM
Press, Washington DC
2 Haseltine WA, Block R, Gilbert W & Weber K (1972)
MSI and MSII made on ribosome in idling step of
pro-tein synthesis Nature 238, 381–384
3 Pedersen FS & Kjeldgaard NO (1977) Analysis of the
relA gene product of Escherichia coli Eur J Biochem 76,
91–97
4 Sy J, Ogawa Y & Lipmann F (1973) Nonribosomal
synthesis of guanosine 5¢, 3¢-polyphosphates by the
ribosomal wash of stringent Escherichia coli Proc Natl Acad Sci USA 70, 2145–2148
5 Haseltine WA & Block R (1973) Synthesis of guanosine tetra- and penta phosphate 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
6 Pedersen FS, Lund E & Kjeldgaard NO (1973) Codon specific tRNA dependent in vitro synthesis of ppGpp and pppGpp Nat New Biol 243, 13–15
7 Wendrich TM, Blaha G, Wilson DN, Marahiel MA & Nierhaus KN (2002) Dissection of the mechanism for the stringent factor RelA Mol Cell 10, 779–788
8 Richter D (1976) Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA Proc Natl Acad Sci USA 73, 707–711
9 Friesen JD, Fiil NP, Parker JM & Haseltine AW (1974)
A new relaxed mutant of Escherichia coli with an altered 50S ribosomal subunit Proc Natl Acad Sci USA
71, 3465–3469
10 Yang X & Ishiguro EE (2001) Involvement of the N ter-minus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli J Bacteriol 183, 6532– 6537
11 Thompson J, Cundliffe E & Stark M (1979) Binding of thiostrepton to a complex of 23S rRNA with ribosomal protein L11 Eur J Biochem 98, 261–265
12 Silverman RH & Atherly AG (1978) Unusual effects of 5a,6-anhydrotetracycline and other tetracyclines Inhibi-tion of guanosine 5¢-diphosphate 3¢-diphosphate meta-bolism, RNA accumulation and other growth-related processes in Escherichia coli Biochim Biophys Acta 518, 267–276
13 Rheinberger HJ, Sternbach H & Nierhaus KH (1981) Three tRNA binding sites on Escherichia coli ribosomes Proc Natl Acad Sci USA 78, 5310–5314
14 Lill R, Robertson JM & Wintermeyer W (1986) Affi-nities of tRNA binding sites of ribosomes from Escheri-chia coli Biochemistry 25, 3245–3255
15 Justesen J, Lund T, Pedersen FS & Kjeldgaard NO (1986) The physiology of stringent factor (ATP:GTP 3¢ diphosphotransferase) in Escherichia coli Biochimie
68, 715–722
16 Howe JG & Hershey WB (1983) Initiation factor and ribosome levels are coordinately controlled in Escheri-chia coligrowing at different rates J Biol Chem 258, 1954–1959
17 Lancaster L, Kiel MC, Kaji A & Noller HF (2002) Orien-tation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing Cell 111, 129–140
18 Moazed D, Stern S & Noller HF (1986) Rapid chemical probing of conformation in 16S ribosomal RNA and 30S ribosomal subunits using primer extension J Mol Biol 187, 399–416