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.

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

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

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

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)

(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.

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

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

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)

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

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

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-

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

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-

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

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

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

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

)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).
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)

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 coli growing 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

from a complete set of recombinant small subunit ribo-
somal proteins. RNA 5, 832–843.
26 Nirenberg M & Leder P (1964) RNA code words and
protein synthesis. The effect of trinucleotides upon the
binding of SRNA to ribosomes. Science 145, 1399–1407.
27 Cate J, Yusupov MM, Yusupova GZ, Earnest TN &
Noller HF (1999) X-ray crystal structures of 70S ribo-
some functional complexes. Science 285 , 2095–2104.
28 Moazed D & Noller HF (1989) Intermediate states in
the movement of transfer RNA in the ribosome. Nature
342, 142–148.
29 Moazed D & Noller HF (1989) Interaction of tRNA
with 23S rRNA in the ribosomal A, P, and E sites. Cell
57, 585–597.
30 Lill R, Robertson JM & Wintermeyer W (1984) tRNA
binding sites of ribosomes from Escherichia coli. Bio-
chemistry 23, 6710–6717.
31 Gao W, Jakubowski H & Goldman E (1995) Evidence
that uncharged tRNA can inhibit a programmed trans-
lational frameshift in Escherichia coli. J Mol Biol 251,
210–216.
32 Howard GA & Gordon J (1976) Stringent factor binds
to Escherichia coli ribosomes only in the presence of
protein L10. FEBS Lett 68, 211–214.
33 Ramagopal S & Davis BD (1974) Localization of the
stringent protein of Escherichia coli on the 50S riboso-
mal subunit. Proc Natl Acad Sci USA 71, 820–824.
34 Goldman E & Jakubowski H (1990) Uncharged tRNA,
protein synthesis, and the bacterial stringent response.
Mol Microbiol 4, 2035–2040.

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