Translation initiation region dependency of translation
initiation in Escherichia coli by IF1 and kasugamycin
Serhiy Surkov
1
, Hanna Nilsson
1
, Louise C. V. Rasmussen
2
, Hans U. Sperling-Petersen
2
and Leif A. Isaksson
1
1 Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden
2 Department of Molecular Biology, Aarhus University, Denmark
Introduction
Translation initiation factor 1 (IF1), encoded by infA,
is a small protein consisting of 71 amino acids in
Escherichia coli. It is essential for cell viability [1], even
though the exact reason for this remains obscure [2,3].
IF1 is highly conserved, and homologous proteins are
present in all three domains of life (IF1 in bacteria,
aIF1A in archeaons, and eIF1A in eukaryotes) [4,5].
IF1 is the smallest of the three initiation factors in
E. coli. During initiation of translation, IF1 binds to
the 30S ribosomal subunit in the A-site region [6,7],
presumably through electrostatic interactions [8]. It
stimulates the action of the other two factors, espe-
cially translation initiation factor 2 (IF2) [9,10]. A
crystal structure analysis of IF1 bound to the 30S
ribosomal subunit shows that the factor is located in
a cleft formed between helix 44, the 530 loop of 16S

ments upstream of the SD sequence and the region between the SD
sequence and the initiation codon are important for the magnitude of this
effect. The data suggest that the wild-type form of IF1 has a translation
initiation region-dependent inhibitory effect on translation initiation. Kasu-
gamycin is an antibiotic that blocks translation initiation. Addition of
kasugamycin to growing wild-type cells increases reporter gene expression
in a very similar way to the altered IF1, suggesting that the infA mutations
and kasugamycin affect some related step in translation initiation. Genetic
knockout of three proteins (YggJ, BipA, and CspA) that are known to
interact with RNA causes partial suppression of the IF1-dependent cold
sensitivity.
Abbreviations
IF1, translation initiation factor 1; IF2, translation initiation factor 2; SD, Shine–Dalgarno; TIR, translation initiation region.
2428 FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS
Several functions are attributed to IF1. It facilitates
IF2-dependent fMet-tRNA binding to the P-site
[10,12,13], probably by stabilizing IF2 binding to the
30S subunit [14], and stimulates the GTPase activity of
IF2 [15]. It also increases binding of mRNA to the ini-
tiation complex in the presence of IF2 [16]. Together
with IF2, it stimulates drop-off of peptidyl-tRNAs
with short polypeptides from 70S ribosomes [17]. In a
recent study, it was shown that IF1 together with IF2
recognizes the formylmethionine moiety of initiator
aminoacyl-tRNA and discriminates against unformy-
lated and deacylated tRNA
f
Met
[3]. IF1 is necessary for
IF2 recycling after subunit joining and GTP hydrolysis

E-site and P-site [32,33]. As no effect of kasugamycin
on mRNA binding to the 30S subunit was shown [34],
it was proposed that the antibiotic effectively distorts
the mRNA structure near the P-site codon, thus pre-
venting efficient fMet-tRNA binding [32,33,35]. Trans-
lation of different mRNAs is affected by kasugamycin
[36], depending on the nature of the nucleotides in
mRNA corresponding to the E-site [32]. Translation of
leaderless mRNA starting directly from AUG is insen-
sitive to kasugamycin action [37].
Expression of a reporter gene is increased in E. coli
strains that carry mutations in the chromosomal infA
(IF1) gene [38]. These mutant strains grow consider-
ably slower than the parental MG1655 strain, and
some of them are cold sensitive for growth. The repor-
ter gene is significantly overexpressed in two cold-sen-
sitive chromosomal IF1 mutant strains or through
addition of kasugamycin to the corresponding wild-
type strain. In this study, we have made an extensive
in vivo analysis of the mRNA sequence composition
that causes such overexpression. We demonstrate simi-
lar effects on TIR-dependent gene expression of the
IF1 mutations and the antibiotic kasugamycin. The
IF1-dependent cold sensitivity is partly suppressed by
elimination by genetic knockout of some other pro-
teins involved in RNA recognition (YggJ, BipA, and
CspA).
Results
Increased gene expression by a mutant form of
IF1

S. Surkov et al. TIR dependence of translation initiation by IF1
FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS 2429
To address this question, we performed a radioactive
double-labeling experiment using the wild-type strain
and the two IF1 cold-sensitive mutants CVR40D and
CVR69L [2]. The pSS101 vector with the 3A¢ and 2A¢
genes was used to study protein A¢ expression in the
strains by gel scanning. Proteins in CVR40D and
CVR69 were labeled with [
3
H]lysine. The MG1655
parental strain was labeled during cultivation with
[
14
C]lysine. Cells were cultivated separately, but each
mutant was pooled with MG1655 when harvested. The
3A¢-encoded and 2A¢-encoded double-labeled proteins
in the mixture were purified and separated on gels.
The
3
H ⁄
14
C isotope ratios were determined for the 2A¢
and 3A¢ gel bands, and compared with the isotope
ratios of the total cellular protein, from the sample
taken before the purification step. As can be seen in
Fig. 1, values for 2A¢ reference gene expression in the
IF1 mutants were quite similar to the values for total
proteins. In contrast, 3A¢ expression was increased by
the IF1 mutations. The increased values for the

as a reference, are slightly higher (1.50- and 1.33-fold,
respectively) than in the wild-type strain. However, no
increase was seen in the 2A¢⁄total protein ratio as a
result of the IF1 mutations, whereas the 3A¢⁄total pro-
tein ratio was increased about two-fold for both of
them (Fig. 1). Preliminary data suggest that overpro-
duction of wild-type IF1 from a multicopy plasmid
does not cause any increase in 3A¢ expression. The
data suggest that the increased 3A¢⁄2A¢ ratio is mostly
the result of changed functionality of the mutant IF1
and not of an altered IF1 level. Because the 3A¢ and
the 2A¢ genes in the pSS101 plasmid are different in
their translation initiation region (TIR) composition,
the data suggest that there are altered functional inter-
actions between the mutated IF1 factor and some
sequence signals in the TIR region. We decided to ana-
lyze these signals.
Effect of the downstream region composition
The influence of different sequences downstream of
AUG on gene expression at the translational level has
been well characterized [41]. Even though IF1 binds to
the A-site of the 30S subunit, different +2 codons do
not cause significantly changed levels of protein expres-
sion in different IF1 mutant strains [38], indicating a
0.0
0.5
1.0
1.5
2.0
2.5

H]lysine. Cultures were pooled together, and the iso-
tope ratios for the total cellular proteins, as well as the 3A¢ and 2A¢
protein bands in PAGE gels, were calculated. The isotope ratios for
the 3A¢ reporter gene and the 2A¢ reference gene are shown in
relation to the isotope ratio for the total cellular protein, which is
taken as unity. TIRs of the 3A¢ and 2A¢ genes in pSS101 are indi-
cated by the SDs in capital letters. The AUG initiation codon is in
underlined capital letters.
TIR dependence of translation initiation by IF1 S. Surkov et al.
2430 FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS
lack of codon specificity. Using CVR40D, we used the
3A¢⁄2A¢ test system to analyze other plasmids with dif-
ferent sequences downstream of the initiation codon in
3A¢ (downstream regions DR-A, DR-B, DR-C, and
DR-D) (Fig. 2) [42] but with a constant upstream
sequence. In CVR40D, the expression levels of these
3A¢ variants with the Shine–Dalgarno (SD)
+
sequence
were elevated, giving an approximately two-fold
increase (Fig. 2), which is similar to the observed value
for the original construct pSS101. This increased gene
expression in CVR40D relative to the parental strain
MG1655 is independent of the composition of the
downstream region.
Influence of the SD sequence and its upstream
sequence
IF1 inhibits the joining of 50S to the preinitiation
complex if the SD sequence in the mRNA is extended.
This effect is not seen for a four base SD [43]. For this

plasmid is toxic for MG1655, which makes correct
evaluation of the expression levels difficult. The reason
for this toxic effect is not clear, and requires further
investigation.
Increased expression was also obtained by the addi-
tion of kasugamycin to MG1655, as discussed below.
The effect of the length of the spacer between a canon-
ical SD
+
sequence and the initiation codon was also
analyzed. As shown in Fig. 3C, the longer spacers,
especially with a 12 base spacer, gave higher gene
expression in the infA mutants.
Comparison of two different reporter gene
systems
Relevant reporter gene sequences were also analyzed
by using the b-galactosidase assay system in MG1655
and CVR40D. For this purpose, the initiation region
of the b-galactosidase gene from the pCMS71 plasmid
[44] was replaced with some of the corresponding
DR-A cuagcuaauaaauuaAGGAGGauuuaaauAUGAAAGCAAUUUUCGUAc
DR-B cuagcuaauaaauuaAGGAGGauuuaaauAUGAGUGAAUCACAAGCCc
DR-C cuagcuaauaaauuaAGGAGGauuuaaauAUGAAAAAGGAGUCGACUc
DR-D cuagcuaauaaauuaAGGAGGauuuaaauAUGACCGAGGGUGUUUCCc
3A′
2A′
DR-A
3A′/2A′ 0.16
0.08
0.370.170.390.210.660.31

3A′/2A′ ratio
3A′/2A′ ratio
3A′/2A′ ratio
Fig. 3. (A) Influence of the SD sequence length on reporter gene expression. Strains and the numbers of bases in SD are indicated. (B) TIR-
dependent reporter gene expression in MG1655, CVR40D and CVR69L or in MG1655 in the presence of 175 lgÆmL
)1
kasugamycin (ksg).
Sequences with different TIRs are shown. The SD region is in bold capital letters, and the initiation codon is in underlined capital letters.
pSS301 and pSS101 represent SD
)
and SD
+
versions of the parental 3A¢ plasmid. pSS201 carries an extension by the ribosome-binding
sequence of ribosomal protein S1 (capital letters). The six bases upstream of SD
+
(capital letters) in pSS103 are from the 2A¢ gene in
pSS101. pSS144 carries an extension of four bases in the spacer downstream of the SD
+
, as compared with pSS103. pSS133 carries three
altered bases in the spacer as compared with pSS101. (C) Influence of the distance (Dis) between the SD sequence and the AUG initiation
codon on reporter gene expression in MG1655, CVR40D and CVR69L. The number of bases forming the distance are indicated. The SD
region is in bold capital letters, and the initiation codon is in underlined capital letters.
TIR dependence of translation initiation by IF1 S. Surkov et al.
2432 FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS
located the antibiotic to the ribosomal tunnel region.
As the effects of the R40D and the R69L mutations
are dependent on the sequence upstream of the initia-
tion codon, we wanted to analyze the effects of these
mutations on growth and reporter gene expression in
comparison with the action of kasugamycin.

concentration of 175 lgÆmL
)1
, as determined by gel
scanning (Fig. 5B). The other bacteriostatic antibiotics
tested (chloramphenicol and tetracycline) markedly
decreased the growth rate of MG1655 cells but did not
influence the 3A¢⁄2A¢ ratio associated with pSS101
(Figs 1 and 5B). Thus, the increased expression caused
by kasugamycin was specific for this antibiotic and
was not caused by the other two antibiotics, which
also inhibit translation. Analysis of reporter gene
expression using plasmid pSS101 showed that the pres-
ence of 50 lgÆmL
)1
kasugamycin in LB medium had
almost no effect on the 3A¢⁄2A¢ ratio in MG1655, but
caused an increased 3A¢⁄2A¢ ratio in CVR40D and
CVR69L (Figs 1 and 5C). At this antibiotic concentra-
tion (50 lgÆmL
)1
), no growth rate reduction was visi-
ble for either MG1655 or the mutant strains. The
3A¢⁄2A¢ ratios were also measured for the different
strains during growth in the presence of 70 or
100 lgÆmL
)1
kasugamycin. At these concentrations,
cells were collected at a D
590
nm of 0.6, before the bac-

3.0
3.5
A
B
CR40D/MG1655 ratio
pSS201, S1 binding site
pSS101, SD
+
pSS301, SD
–
pSS103, mutated sequence upstream of SD
pSS133, mutated spacer
pSS144, spacer extended by 4 bases
Fig. 4. Comparison of gene expression measurements using two
different reporter gene systems. The ratios of gene expression val-
ues (3A¢⁄2A¢) in CVR40D and MG1655 as measured by the b-galac-
tosidase (A) and protein A¢ (B) assays are shown. The plasmids
used for the comparison are indicated, and their TIR sequences are
given in Fig. 3. Spacer refers to the sequence between the initia-
tion codon AUG and the SD region.
S. Surkov et al. TIR dependence of translation initiation by IF1
FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS 2433
gene knockouts by P1 transduction into CVR40D and
kanamycin selection [45]. For screening, we chose 69
nonessential genes that are known to be associated
with ribosome function or maturation as well as cold
shock response. The double mutant strains were tested
for growth on LB plates at 18 °C. It was found that
inactivation of bipA, yggJ or cspA partly suppressed
the cold-sensitive phenotype of the IF1 mutant

for the 2A¢⁄total protein ratio as compared with the
Growth in the presence of
kasugamycin
0
1
2
3
4
5
6
A
0 200 400 600
Time (min)
D
590
MG1655
MG1655, ksg 70 µg·mL
–1
MG1655, ksg 140 µg·mL
–1
CVR40D
CVR40D, ksg 70 µg·mL
–1
CVR40D, ksg 140 µg·mL
–1
0.0
0.1
0.2
0.3
0.4

MG1655
CVR40D
CVR69L
3A′/2A′ ratio
B
C
Fig. 5. (A) Growth in LB medium in the presence of kasugamycin
(ksg). Filled symbols represent MG1655 and open symbols repre-
sent the mutant CVR40D. (B) Reporter gene expression in MG1655
in the presence of kasugamycin (ksg) or other antibiotics. The plas-
mid was pSS101, as described in Fig. 1. Cam, chloramphenicol;
Tet, tetracycline. (C) Reporter gene expression (pSS101) in
MG1655 or in CVR40D and CVR69L in the presence of different
kasugamycin concentrations.
1
3
2
4
Fig. 6. Growth of CVR40D and derivatives. The indicated strains
were incubated on an LB plate at 18 °C for 4 days. 1, CVR40D; 2,
CVR40D DcspA; 3, CVR40D DbipA; 4, CVR40D DyggJ.
TIR dependence of translation initiation by IF1 S. Surkov et al.
2434 FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS
wild-type strain, whereas the 3A¢⁄total protein ratio is
increased about two-fold. Taken together, the data
suggest that most of the increase in the 3A¢⁄2A¢ ratio
in the mutants is the result of changed functionality of
IF1 and not overproduction of mutant IF1.
Normally, one would expect a mutationally altered
protein to show lowered, not increased, activity. Our

either increased or decreased, also suggesting a differ-
ent sensitivity to the antibiotic (not shown).
IF1 binds to different synthetic polynucleotides in
solution, and it contains an oligomer-binding motif
with high homology to the RNA-binding domains of
ribosomal protein S1 and polynucleotide phosphory-
lase [23–25]. The crystal structure of IF1 in complex
with the 30S ribosomal subunit suggests that IF1 could
directly contact mRNA nucleotides in the ribosomal
A-site [11]. IF1 affects the conformation of 16S rRNA,
causing a movement of helix 44 and a global confor-
mational change in the 30S subunit. This is visible as
the movement of the head of the subunit towards the
body and flipping of bases A1492 and A1493. This
flipping has been shown to constitute an important
part of the quality control signaling during tRNA or
RF factor recognition of the A-site [11]. The R40D
mutant described here is altered in its binding pocket
for the base A1493 [2]. This suggests a direct interac-
tion effect of the IF1 mutation. As IF1 influences the
splicing of a group I intron in vivo and in vitro, and
influences RNA annealing in vitro, the factor has an
RNA chaperone activity [26]. It is conceivable that the
mutant forms of IF1 are less capable of setting the
intricate balance between favoring and disfavoring
higher RNA structures, in the rRNA, mRNA or both,
that are necessary for translational initiation. As a
result, the initiation machinery could be biased such
that the initiation conformation in the mutants is too
high, giving the observed decreased mutant growth

partly suppressed by inactivation of yggJ, bipA,or
cspA. YggJ is a methylase that specifically modifies uri-
dine 1498 of the 16S rRNA. This base is located in the
mRNA channel upstream of AUG. The residue
directly contacts the kasugamycin molecule in the
X-ray structure [32]. It appears likely that it influences
the TIR selection or AUG adjustment specificity of
IF1. BipA is a protein that disrupts SD–antiSD
interactions in some mRNAs during the first steps of
S. Surkov et al. TIR dependence of translation initiation by IF1
FEBS Journal 277 (2010) 2428–2439 ª 2010 The Authors Journal compilation ª 2010 FEBS 2435
translation [47]. Both of these two gene products are
connected to the TIR-specific response of the IF1
mutants, suggesting that the imbalance in the mRNA
translation is the primary reason for the cold sensitiv-
ity of the infA mutants studied here. It is conceivable
that elimination of yggJ or bipA could partially com-
pensate for the enhanced translation initiation of
mRNAs that is observed in the IF1 mutant strains.
CspA facilitates translation initiation at low temper-
atures by melting the mRNA secondary structure [39].
Cold shock stimulates expression of IF1 at the levels
of both transcription and translation [28,29]. Thus,
cold shock constitutes a common denominator for
cspA and infA. The compensation of the cold sensitiv-
ity of IF1 mutant strains by the inactivation of cspA is
another functional link to IF1.
In view of the finding that IF1 has RNA chaperone
activity [26], the effects of the elimination of BipA,
YggJ and CspA are conceivable, as all of them influ-

were grown in M9 minimal medium supplemented with
ampicillin and all amino acids except lysine. 50 lL of the
overnight cell culture were inoculated into 5 mL of the same
medium, and they were grown with intensive aeration to a
D
590 nm
of 0.2. At this point, 75 lL of a lysine solution
labeled with
14
C (50 lCiÆmL
)1
) was added to MG1655 cells,
and 35 lL of lysine solution labeled with
3
H (1 mCiÆmL
)1
)
was added to CVR40D and CVR69L cells. The cells were
grown to a D
590 nm
of 0.6. Then, cold lysine was added to the
final concentration of 0.25 mm, and the cultures were grown
for an additional 20 min at 37 °C for CVR40D and at 30 °C
for CVR69L. The difference in the growth temperature is
due to different cold sensitivities of the strains. CVR40D
shows a decreased growth rate, down to 50%, at 37 °C.
CVR69L is grown at 30 °C to obtain a similar decrease in
growth rate. The cultures were cooled, and MG1655 cells
were combined with CVR40D or CRV69L cells. The com-
bined cells were washed, harvested, and processed for pro-

14
C-labeled
cell cultures into 10% trichloroacetic acid with 1% casamino
acids. The precipitates were washed with 5% trichloroacetic
acid containing 0.1% casamino acids, whereafter the filters
were dried and radioactivity was measured in a scintillation
counter. Alternatively, the pooled
3
H-labeled and
14
C-labeled
cell lysate was loaded onto a polyacrylamide gel, and all of
the resulting protein bands in one lane were excised together
and counted as described above. Both methods gave similar
3
H ⁄
14
C ratios. Similar PAGE experiments were performed
with MG1655 cells expressing 3A¢ and 2A¢ proteins encoded
by the pSS101 plasmid in the presence or absence of
175 lgÆmL
)1
kasugamycin.
Protein A¢ assay
The protein A¢ reporter system has been extensively
described [49]. Briefly, a plasmid carries two genes under
the control of identical trc promoters. Both proteins are
composed of identical A¢ building blocks derived from the
IgG-binding domain (also known as the Z domain) of
Staphylococcus aureus protein A. One gene, encoding three

the trc promoter is leaky, giving significant expression even
in the absence of induction. b-Galactosidase acivity of the
lysed uninduced cells was then determined as described pre-
viously [50].
Plasmids and strains
All plasmids used are based on the pHN109 vector [44],
which carries the 2A¢ internal control gene and the 3A¢ test
gene. The different initiation regions in the 3A¢ test gene in
the plasmids used are shown in the corresponding figures.
P1 transduction was performed according to Miller [51].
The MG1655 strain (F
)
, ilvG, rfb-50, rph) was used as a
wild-type reference strain. Its derivatives CVR40D and
CVR69L have the same genotype except for the R40D
or R69L mutations, respectively, in the infA gene on the
chromosome [2].
Growth curves
Thirty microliter volumes of overnight cultures were inocu-
lated into 3 mL of fresh LB. Cells were grown with intensive
shaking to a D
590 nm
of 0.6, and then diluted in LB to obtain
a D
590 nm
of 0.05 in the presence or absence of kasugamycin;
this was followed by measurements of bacterial growth.
Cold sensitivity complementation test
The gene deletions tested were transferred from the KEIO
strain collection [45] by P1 transduction into CVR40D on

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