AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo. Proc Natl Acad Sci USA 84: 4022

Institute of Physical and Chemical Biology, Lutetia Parisorum, Île-de-France, France
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/1987; 84(12):4022-5. DOI: 10.1073/pnas.84.12.4022
Source: PubMed
ABSTRACT
We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Full-text

Available from: Mathias Springer
Proc.
Nati.
Acad.
Sci.
USA
Vol.
84,
pp.
4022-4025,
June
1987
Biochemistry
AUU-to-AUG
mutation
in
the
initiator
codon
of
the
translation
initiation
factor
IF3
abolishes
translational
autocontrol
of
its
own
gene
(infC)
in
vivo
(protein
synthesis
control/site-specific
mutagenesis)
J.
SCOTT
BUTLER*,
MATHIAS
SPRINGERt,
AND
MARIANNE
GRUNBERG-MANAGOt
*Department
of
Biochemistry,
University
of
Rochester
Medical
Center,
601
Elmwood
Avenue,
Rochester,
NY
14642;
and
tInstitut
de
Biologie
Physico-Chimique,
13,
rue
Pierre
et
Marie
Curie,
Paris
75005,
France
Contributed
by
Marianne
Grunberg-Manago,
February
20,
1987
ABSTRACT
We
previously
showed
that
Escherichia
coli
translation
initiation
factor
IF3
regulates
the
expression
of
its
own
gene
infC
at
the
translational
level
in
vivo.
Here
we
create
two
alterations
in
the
iniC
gene
and
test
their
effects
on
translational
autocontrol
of
infC
expression
in
vivo
by
measur-
ing
13-galactosidase
activity
expressed
from
infC-lacZ
gene
fusions
under
conditions
of
up
to
4-fold
derepression
or
3-fold
repression
of
infC
expression.
Replacement
of
the
infC
pro-
moter
with
the
trp
promoter
deletes
120
nucleotides
of
the
infC
mRNA
5'
to
the
translation
initiation
site
without
affecting
autogenous
translational
control.
Mutation
of
the
unusual
AUU
initiator
codon
of
infC
to
the
more
common
AUG
initiator
codon
abolishes
translation
initiation
factor
IF3-dependent
repression
and
derepression
of
infC
expression
in
vivo.
These
results
establish
the
AUU
initiator
codon
of
infC
as
an
essential
cis-acting
element
in
autogenous
translational
control
of
trans-
lation
initiation
factor
IF3
expression
in
vivo.
The
initiation
of
protein
synthesis
in
Escherichia
coli
requires
the
activity
of
three
protein
factors
(IF1,
IF2,
and
IF3)
(for
reviews,
see
refs.
1-3).
One
of
these
initiation
factors,
IF3,
binds
to
the
30S
ribosomal
subunit
and
shifts
the
equilibrium
between
70S
ribosomes
and
their
50S
and
30S
ribosomal
subunits
in
favor
of
the
free
subunits,
thus
enhancing
the
availability
of
30S
subunits
on
which
protein
synthesis
initiation
begins.
In
addition,
IF3
may
play
a
direct
role
in
the
binding
of
mRNA
to
the
30S
ribosome.
The
cellular
levels
of
the
protein
synthesis
initiation
fac-
tors,
like
other
protein
synthesis
components,
increase
with
increases
in
cellular
growth
rate
(4),
suggesting
a
shared
system
of
genetic
regulation.
However,
the
genes
for
each
of
the
factors
do
not
map
adjacent
to
one
another,
implying
that
coordinate
control
of
IF
gene
expression
requires
separate,
but
not
necessarily
independent,
control
elements.
The
gene
for
IF2,
infB,
lies
at
69
min
on
the
E.
coli
genetic
map,
and
it
appears
that
the
product
of
the
adjacent
gene,
NusA
protein,
regulates
both
infB
and
nusA
gene
expression
at
the
transcriptional
level
in
vivo
(5,
6).
The
infC
gene
for
IF3
maps
at
38
min
on
the
E.
coli
chromosome
(7),
adjacent
to
other
genes
involved
in
protein
synthesis
(8).
The
genetic
organi-
zation
of
infC
is
very
unusual:
(i)
infC
is
unique
in
initiating
translation
at
an
AUU
codon
(9),
(ii)
the
infC
AUU
initiator
codon
lies
only
three
nucleotides
3'
to
the
stop
codon
of
thrS,
the
gene
for
threonyl-tRNA
ligase
(10),
and
(iii)
transcription
of
the
majority
of
infC
mRNAs
initiates
at
a
promoter,
pO',
inside
the
thrS
coding
sequence
and
182
base
pairs
upstream
of
the
infC
initiator
AUU
(11).
Our
recent
experiments
demonstrated
that
IF3
regulates
expression
of
its
own
gene
at
the
translational
level
in
vivo
(11).
First,
infC
mutant
strains
with
elevated
expression
of
structurally
altered
IF3
do
not
have
corresponding
increases
in
infC
mRNA
synthesis
rates.
Second,
infC
mutant
alleles
cause
derepression
of
infC-lacZ
gene
fusions
(translation
from
the
translation
initiation
site
of
infC)
but
not
infC-lacZ
operon
fusions
(translation
from
the
translation
initiation
site
of
lacZ).
Third,
a
cellular
excess
of
IF3
supplied
in
trans
from
a
multicopy
plasmid
represses
expression
of
an
infC-lacZ
gene
fusion
without
affecting
the
rate
of
synthesis
of
hybrid
infC-lacZ
mRNA.
In
the
present
work
we
tested
whether
the
unusual
AUU
initiator
codon
plays
a
role
in
infC
translational
autoregula-
tion.
The
results
clearly
show
that
a
single
mutation
of
AUU-to-AUG
abolishes
autogenous
translational
control
of
infC
expression.
This
result
is
discussed
in
light
of
a
theo-
retical
analysis
of
the
infC
system
by
Gold
et
al.
(12)
that
predicted
the
infC
AUU
initiator
codon
to
be
an
essential
element
in
the
autoregulatory
system.
MATERIALS
AND
METHODS
The
E.
coli
K-12
strains
used
here
were
as
described
(11).
Genetic
techniques
and
P3-galactosidase
measurements
were
as
described
by
Miller
(13).
Molecular
cloning
techniques
were
as
described
by
Maniatis
et
al.
(14).
Selection
and
screening
of
X
monolysogens
were
as
described
(15).
Mea-
surements
of
infC-lacZ
mRNA
levels
were
made
as
described
(11)
with
details
given
in
the
legend
to
Table
3.
Construction
of
M13mpl9TSX3.
Our
first
step
in
in
vitro
mutagenesis
of
infC
was
to
clone
the
HindIII-Sal
I,
trpPO-
containing
fragment
of
pDR720
(16)
into
the
same
sites
in
M13mpl9
to
yield
the
Lac-
derivative
M13mpl9ptrp4.
Next,
the
promoterless
Sal
I-Sst
II
fragment
of
pUSX15
(11)
containing
the
translation
initiation
site
and
the
first
54
codons
of
infC
was
inserted
into
the
same
sites
between
trpPO
and
lacZ
on
M13mpl9ptrp4
to
make
the
Lac'
deriv-
ative
M13mp19TSX3.
Oligonucleotide
Directed
Site
Specific
Mutagenesis.
We
used
the
deoxyoligonucleotide
5'
TCCGCCTTTCATACCTTA
3'
provided
by
B.
Ehresmann
(Institut
de
Biologie
Moleculaire
et
Cellulaire
du
Centre
National
de
la
Recherche
Scientifique,
Laboratoire
de
Biochimie,
Strasbourg,
France)
to
mutate
the
infC
AUU
initiator
codon
to
AUG
using
the
method
of
Kunkel
(17).
The
nucleotide
sequence
of
the
resulting
mutant
M13mpl9TSG2
and
the
parent
M13mp19TSX3
was
deter-
mined
(18)
by
sequencing
across
the
M13mpl9
polylinker
at
the
infC-lacZ
fusion
all
the
way
to
the
EcoRI
site
just
5'
to
trpPO.
The
change
of
AUU-to-AUG
is
the
only
difference
between
the
two
sequences.
Transfer
of
Wild-Type
and
Mutant
infC-lacZ
Fusions
to
X.
To
allow
study
of
our
infC-lacZ
gene
fusions
as
single
copies
Abbreviation:
IF,
translational
initiation
factor.
4022
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
Page 1
Proc.
Nati.
Acad.
Sci.
USA
84
(1987)
4023
in
vivo,
we
constructed
a
X
bacteriophage
carrying
all
of
the
lac
operon
except
lacI,
the
promoter,
the
translation
initia-
tion
site,
and
the
first
seven
amino
acids
of
lacZ.
The
EcoRI-Sst
II
lac
fragment
of
pNM482
(19)
was
inserted
between
the
left
arm,
up
to
the
Sst
II
site
of
XNM540
(20),
and
the
right
arm,
from
the
far
right
EcoRI
site
of
XSCX16
(11).
After
in
vitro
packaging
we
characterized
by
restriction
analysis
the
c1857,
Lac-
phage
XNNS4.
The
EcoRI
fragments
containing
the
trpPO-infC
fusion
of
M13mp19TSX3
and
its
mutant
derivative
M13mp19TSG2
were
inserted
separately
into
the
EcoRI
site
in
front
of
lac
in
XNNS4,
creating
an
in-frame
gene
fusion
between
infC
and
lacZ.
After
in
vitro
packaging
and
screening
of
Lac'
X
phage,
we
characterized
by
restriction
analysis
the
respective
de-
rivatives
XTSX31
(wild
type,
AUU)
and
XTSG25
(mutant,
AUG)
(Fig.
1).
RESULTS
Replacement
of
the
inIC
Promoter
with
the
tp
Promoter
Does
Not
Affect
Translational
Autoregulation
of
iniC
Expres-
sion.
We
replaced
the
infC
promoter
pO'
with
the
controllable
trp
promoter
because
changing
the
infC
initiator
AUU
to
AUG
in
the
presence
pO'
resulted
in
apparent
instability
of
infC-lacZ
gene
fusions
cloned
in
X
(J.S.B.,
unpublished
results),
possibly
due
to
lethal
levels
of
hybrid
IF3-3-
galactosidase
expression.
The
data
in
Table
1
show
that
although
IF3-p-galactosidase
expression
decreases
from
519
to
93
units
when
the
infC
pO'
promoter
is
replaced
with
the
trp
promoter,
the
level
of
derepression
from
the
fusions
is
WtC
ClacZ
att
pO'
AUU
N
trpPO
infC
lacZ
J
XSCX
16
Y
A
att
ptrp
AUU
trpPO
infC''lacZ
Y
A
att
ptrp
AUG
FIG.
1.
Physical
maps
of
the
X
bacteriophage
canr
gene
fusions.
The
X
genomes
are
abbreviated
and
direction
opposite
to
normal.
Boxes
above
the
line
re
DNA.
Open
boxes
below
the
line
represent
X
DNA.
)
or
after
a
gene
means
that
it
is
incomplete
on
that
sid
represent
mRNA
transcripts
and
are
preceded
by
a
their
promoter.
The
nucleotide
triplets
represent
t
initiation
codons
and
the
approximate
positions
in
D
where
infC-lacZ
protein
synthesis
initiates.
Y,
lacY
gene;
and
J,
J
gene.
I
I
J
XTSX
31
exactly
the
same:
the
derepression
factor
is
about
four
in
an
infCl9
background
and
about
two
in
an
infC37
background
whatever
promoter
is
used
to
express
the
infC-lacZ
gene
fusion.
Similarly,
the
data
in
Table
2
show
that
an
excess
of
IF3
produced
from
an
infC-carrying,
multicopy
plasmid
represses
IF3-l3-galactosidase
expression
almost
3-fold
with
either
the
trp
or
the
infC
pO'
promoter.
We
showed
previ-
ously
that
such
repression
of
infC
pO'
gene
fusions
occurs
without
changes
in
the
synthesis
rate
of
infC-lacZ
mRNA
(11),
and
the
data
in
Table
3
show
that,
using
the
trp
promoter,
the
derepression
seen
in
an
infCJ9
background
occurs
without
a
significant
change
in
the
rate
of
synthesis
of
infC-lacZ
mRNA.
These
results
demonstrate
that
transla-
tional
autoregulation
of
infC
expression
occurs
independent
of
the
nature
of
the
promoter
expressing
the
gene
and
thereby
confirm
our
previous
experimental
results
showing
autog-
enous
translational
control
of
infC
expression
in
vivo
(11).
Changing
the
infC
AUU
Initiator
Codon
to
AUG
Abolishes
Translational
Autoregulation
of infC
Expression
in
Vivo.
The
unusual
usage
of
an
AUU
initiator
codon
in
translation
of
infC
(9)
and
the
fact
that
IF3
regulates
its
own
gene
expres-
sion
at
the
level
of
translation
(11)
suggest
that
the
AUU
codon
may
play
a
role
in
the
autoregulatory
system
(12).
To
test
this
hypothesis
we
changed
the
infC
initiator
AUU
to
the
more
common
initiator
AUG
and
tested
the
ability
of
IF3
to
control
its
own
gene
expression
by
monitoring
the
levels
of
IF3-f3-galactosidase
and
infC-lacZ
mRNA
expressed
from
infC-lacZ
gene
fusions
under
conditions
normally
causing
derepression
or
repression
of
infC
expression
in
vivo.
Mu-
tation
of
the
AUU
initiator
codon
of
infC
has
two
interesting
effects
on
infC
expression
in
vivo.
First,
initiation
of
trans-
lation
with
AUG
(XTSG25)
instead
of
AUU
(XTSX31)
in-
creases
the
expression
of
an
infC-lacZ
gene
fusion
10-fold
in
a
wild-type
background
(Table
1)
without
a
comparable
increase
in
infC-lacZ
mRNA
levels
(Table
3).
These
results
indicate
that
the
lower
level
of
expression
from
the
fusion
carried
by
XTSX31
relative
to
XTSG25
is
due
to
a
transla-
tional,
instead
of
transcriptional,
phenomenon.
Second,
and
most
dramatically,
the
mutation
of
AUU
to
AUG
completely
abolishes
translational
autoregulation
of
infC
expression.
Neither
infC
mutant
alleles
(Table
1)
nor
over-production
of
IF3
in
trans
(Table
2)
affects
AUG-initiated
translation
of
the
infC-lacZ
gene
fusion
carried
by
XTSG25,
whereas
its
AUU
counterpart
XTSX31
is
normally
regulated.
We
conclude,
therefore,
that
the
infC
AUU
initiator
codon
is
a
necessary
cis-acting
element
in
the
translational
autoregulation
of
infC
expression
in
vivo.
DISCUSSION
The
experimental
results
presented
here
identify
the
unusual
initiator
AUU
codon
of
infC
as
a
necessary
cis-acting
element
in
autogenous
translational
control
of
infC
expression
in
vivo.
Replacement
of
the
major
promoter
pO'
of
infC
by
the
trp
promoter
has
no
effect
on
autogenous
translational
control
of
infC
expression,
confirming
our
previous
conclusion
that
IF3
controls
its
own
synthesis
at
the
translational
level
in
vivo
J
(11).
The
replacement
also
deletes
the
first
120
nucleotides
of
the
leader
of
the
infC
mRNA
without
affecting
regulation
of
)xTSG25
infC
expression,
indicating
that
information
required
for
translational
autocontrol
lies
in
a
region
62
nucleotides
5'
to
rying
infC-lacZ
the
initiator
AUU.
Because
each
of
the
gene
fusions
used
drawn
in
the
here
contains
the
first
54
codons
of
infC
fused
to
lacZ
we
present
E.
coli
cannot
exclude
the
possibility
that
control
elements
lie
in
the
k
prime
before
159
nucleotides
3'
to
the
AUU
codon.
le.
The
arrows
The
most
important
result
of
our
experiments
is
that
a
single
he
translation
nucleotide
mutation
changing
the
wild-type
AUU
initiator
'NA
and
RNA
codon
to
AUG
eliminates
autogenous
translational
control
of
gene;
A,
lacA
infC
expression.
This
mutation
causes
the
loss
of
derepression
of
infiC
expression
by
infC
mutant
alleles
(Table
1)
and
the
loss
Biochemistry:
Butler
et
al.
Page 2
Proc.
Natl.
Acad.
Sci.
USA
84
(1987)
Table
1.
Effect
of
infC
alleles
on
infC-lacZ
expression
from
various
X
bacteriophage
3-Galactosidase
expression,
units
Derepression
factor
Gene
fusion
Wild
type
infCI9
infC37
infCl9/wt
infC37/wt
XSCX16
(wild-type
infC
promoter,
AUU
initiator
codon)
519
±
20
1942
±
239
1020
±
20
3.7
1.9
XTSX31
(trp
promoter,
AUU
initiator
codon)
93
±
6
358
±
8
179
±
2
3.8
1.9
XTSG25
(trp
promoter,
mutant
AUG
codon)
1037
±
56
976
±
27
899
±
6
0.94
0.87
Measurements
were
made
on
monolysogenic,
AWac
strains
IBPC5311
(wild
type),
IBPC5231
(infCl9),
and
IBPC5251
(infC37)
(11)
growing
in
exponential
phase
at
30'C
in
4-morpholinepropanesulfonic
acid
(Mops)-glucose
culture
media
(21)
supplemented
with
arginine
at
0.05
mg/ml.
The
doubling
times
for
the
strains
are
120
min
each.
The
values
are
the
average
of
five
or
six
measurements
±
SD
and
are
expressed
per
A650
unit
of
bacteria
as
described
by
Miller
(13).
of
repression
of
infC
expression
by
increased
cellular
levels
of
IF3
(Table
2)
without
a
significant
increase
in
infC-lacZ
mRNA
levels
(Table
3).
It
is
not
likely
that
the
increased
translational
efficiency
caused
by
mutation
of
AUU-to-AUG
can,
alone,
account
for
loss
of
translational
autocontrol
because
excess
IF3
represses,
2.5-fold,
comparable
levels
of
IF3-f3-galactosidase
expression
from
the
wild-type
fusion
on
XSCX16
(Table
2).
Instead,
the
AUU
initiator
codon
must
itself
specify
recognition
by
the
system
governing
translational
autocontrol
of
infC
expression,
and
its
mutation
to
AUG
results,
most
likely,
in
a
combination
of
derepression
of
translation
initiation
due
to
loss
of
autocontrol
and
an
increase
in
efficiency
of
translation
initiation
from
AUG
instead
of
AUU.
Our
earlier
experimental
results
showing
autogenous
trans-
lational
control
of
infC
expression
(11)
and
the
present
results
showing
its
dependency
on
an
AUU
initiator
codon
confirm
two
predictions
of
a
theoretically
based
model
proposed
by
Gold
et
al.
(12).
In
their
model
infC
mRNA
is
translated
in
an
IF3-independent
manner
due
to
the
abnormal
AUU
initiator
codon
and
other
unusual
features
of
the
translation
initiation
site
of
infC.
The
model
predicts
that
when
the
IF3
level
is
low
relative
to
the
number
of
30S
ribosomal
subunits,
IF3-
independent
translation
of
infC
mRNA
will
be
preferred
to
IF3-dependent
translation
of
other
cellular
mRNAs-causing
a
relative
increase
in
IF3
levels.
The
Gold
et
al.
model
also
predicts
twenty-nucleotide
base
pairing
interactions
between
the
translation
initiation
site
of
infC
mRNA
and
various
domains
of
the
16S
RNA
that
specify
infC
mRNA-30S
ribosomal
subunit
interaction
in
the
proposed
IF3-indepen-
dent
mode.
In
this
respect,
it
is
somewhat
surprising
that
a
single
mutation
of
U-to-G,
changing
only
a
single
base
pair,
should
result
in
complete
loss
of
translational
autoregulation.
It
is,
however,
possible
that
the
AUU-to-AUG
change
is
enough
to
favor
translation
of
the
mutated
infC
mRNA
in
the
IF3-dependent
mode,
which
should,
if
the
model
is
correct,
cause
loss
of
autoregulation
of
IF3
synthesis.
The
negative
autoregulation
of
infC
translation
can
be
explained
alternately
by
a
more
conventional
model
whereby
IF3
binds
to
an
operator
site
covering
the
translation
initia-
tion
site
on
the
infC
mRNA.
In
this
case,
the
third
base
of
the
AUU
initiator
codon
should
be
essential
to
the
binding
of
IF3
to
the
translational
operator.
Chemical
crosslinking
experi-
ments
(22)
and
assays
showing
binding
of
IF3
to
the
3'
end
of
the
16S
RNA
(23)
suggest
that
IF3
may
interact
directly
with
sites
on
the
16S
RNA
during
translation
initiation.
Some
nucleotide
sequence
homology
exists
between
these
sites
and
the
translation
initiation
site
of
infC
(9,
22,
23)-yet
these
homologies
do
not
include
the
infC
initiator
AUU
codon.
Such
nucleotide
sequence
homologies
might
participate
in
secondary
structures
recognized
by
IF3
and
destabilized
by
mutation
of
AUU-to-AUG.
However,
examination
of
the
nucleotide
sequence
of
the
portion
of
infC
shown
here
to
specify
autocontrol
does
not
reveal
significant
secondary
structure
involving
the
translation
initiation
site.
Lack
of
apparent
homology
between
the
16S
RNA
and
infC
mRNA
does
not
rule
out
the
existence
of
a
functional
competition
between
the
RNAs
for
IF3.
We
see
little
reason
at
this
time,
however,
to
favor
a
classical
IF3-mRNA
interaction
model
over
the
more
indirect
model
of
Gold
et
al.
A
growing
number
of
proteins
control
their
own
gene
expression
at
the
translational
level
in
vivo.
These
include
genes
for
ribosomal
proteins
(24,
25),
the
gene
32
protein
(26,
Table
2.
Effect
of
a
cellular
excess
of
IF3
on
infC-lacZ
expression
from
various
X
bacteriophage
3-Galactosidase
expression,
units
pBR322
pSB1
Repression
factor
Gene
fusion
(vector
plasmid)
(infC
plasmid)
pSB1/pBR322
XSCX16
(wild-type
infC
promoter,
wild-type
infC
AUU
initiator
codon)
981
+
86
363
±
80
0.37
XTSX31
(trp
promoter,
wild-type
infC
AUU
initiator
codon)
135
±
10
57
±
2
0.42
XTSG25
(trp
promoter,
mutant
infC
AUG
initiator
codon)
790
±
83
762
±
65
0.97
The
strains
and
techniques
are
described
in
the
legend
of
Table
1
except
that
4-morpholinepropane-
sulfonic
(Mops)-glucose
culture
media
was
supplemented
with
all
the
amino
acids
(21)
except
tryptophan;
ampicillin
was
added
to
0.1
mg/ml
every
2
hr
during
growth;
and
the
doubling
time
for
the
strains
was
90
min.
4024
Biochemistry:
Butler
et
al.
Page 3
Proc.
Natl.
Acad.
Sci.
USA
84
(1987)
4025
Table
3.
Cellular
levels
of
infC-lacZ
mRNA
expressed
from
infC-lacZ
gene
fusions
RNA
Percent
Input
RNA,
bound,
bound,
Strain
cpm
x
l0-4
cpm
%
x
102
IBPC5311XTSX31
(wild-type
strain,
gene
fusion
with
wild-type
AUU
initiator
codon)
103
91
±
17
0.9
±
0.2
IBPC5311XTSG25
(wild-type
strain,
gene
fusion
with
mutant
AUG
initiator
codon)
58
90
±
24
1.5
±
0.4
IBPC5231XTSX31
(infCJ9
strain,
gene
fusion
with
wild-type
AUU
initiator
codon)
166
170
±
2
1.0
±
0.01
The
strains
were
grown
as
described
in
the
legend
of
Table
1.
The
[3H]uridine-labeled
RNA
was
isolated
and
hybridized
to
an
excess
of
the
lacZ-specific
probe
M13mp81acl4
as
described
(11).
Background
hybridization
to
single-stranded
M13mp8
was
<25%
of
the
values
in
column
3
and
has
been
subtracted
from
them.
27)
and
the
RegA
protein
(28)
of
bacteriophage
T4,
and
threonyl-tRNA
ligase
(15,
29).
In
all
of
these
examples
(RegA
being
a
possible
exception)
the
gene
products
made
by
and
controlling
each
gene
interact
functionally,
in
a
well-defined
manner,
with
nucleic
acids.
The
ability
of
the
protein
to
control
translation
is
thought
to
depend
upon
its
binding
to
a
region
on
its
own
mRNA
that
shares
some
homology
with
its
primary
nucleic
acid
substrate.
For
example,
mutations
on
the
thrS
mRNA
disrupting
translational
repression
by
thre-
onyl-tRNA
ligase
lie
in
an
area
of
the
mRNA
adjacent
to
the
ribosome
binding
site
showing
striking
homologies
in
se-
quence
and
structure
to
threonine
isoacceptor
tRNAs
(30).
IF3
differs
from
these
examples
because,
although
it
interacts
with
a
variety
of
RNAs,
it
does
so
without
any
apparent
nucleotide
sequence
specificity
(23,
31,
32).
Nevertheless,
IF3
may
control
its
own
translation
simply
by
binding
to
its
AUU
codon,
thereby
blocking
the
access
of
30S
ribosomes.
Such
a
model,
together
with
the
results
presented
here,
suggests
that
IF3
should
have
a
measurable
difference
in
binding
affinity
for
infC
mRNAs
containing
an
AUU
initiator
codon
or
a
AUG
initiator
codon.
Translation
initiation
of
E.
coli
mRNAs
occurs
at
specific
sites
characterized
by
a
certain
number
of
determinants
whose
importance
depends
upon
their
spatial
organization
(33).
One
of
these
determinants
is
the
translation
initiation
codon
to
which
initiator
fMet-tRNA
binds
on
the
30S
ribo-
somal
subunit.
The
translation
initiation
codon
is
generally
AUG,
but
E.
coli
initiator
tRNA
can
also
recognize
GUG
and
exceptionally
AUA
and
UUG
(33).
Surprisingly,
IF3,
which
is
essential
for
efficient
translation
initiation
complex
forma-
tion
in
vitro,
has
a
unique
AUU
initiator
codon.
The
results
reported
here
show
that
this
exception
is
not
fortuitous,
but
is
necessary
for
the
controlled
synthesis
of
IF3
itself.
The
authors
are
grateful
to
Terry
Platt,
for
reading
the
manuscript
and
for
generously
providing
laboratory
facilities
where
this
work
was
completed.
J.S.B.
was
supported
by
the
National
Institutes