FEMS MicrobiologyLetters 84 (1991) 325-330
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
325
FEMSLE 04709
Initiation of translation at AUC, A U A and A U U codons
in Escherichia coli
Alicia R o m e r o a n d P e d r o Garc~a
Unidad de Gendtica Bacteriana, Centro de Investigaciones Bioldgicas, Consejo Superior de Investigaciones Cienfffieas, Madrid, Spain
Received 16 September 1991
Accepted 19 September 1991
Key words: Translational efficiency; Initiation codons; Murein hydrolase; Escherichia coli
1. S U M M A R Y
A truncated form of the H B L murein hydrolase, encoded by the t e m p e r a t e bacteriophage
HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using A U C as start
codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed
mutagenesis, we have changed this A U C codon
into AUA, A U U and A U G codons. The relative
translation efficiencies for these triplets were
about 5% for A U C and A U U and 7.5% for A U A
c o m p a r e d to that of A U G codon. In the same
gene arrangement E. coli /3-galactosidase was
also translated at moderate efficiency using A U C
as initiator.
2. I N T R O D U C T I O N
The molecular mechanisms involved in the rib o s o m e - m e d i a t e d synthesis of proteins with
Correspondence to: P. Garcla, Unidad de Gen&ica Bacteriana, Centro de Investigaciones Biol6gicas, CSIC, Vel~zquez •
144, E-28006 Madrid, Spain.
m R N A as template had been highly conserved in
all organisms studied [1]. It is well established
that the two most important sequences in m R N A
that are known to determine the efficiency of
translation initiation are the ribosome binding
site (RBS) and the translation start codon. The
available data indicate that RBS strengths vary
over a wide range and mutations in the RBS that
diminish its interaction with the 16S r R N A reduce or block initiation unless compensating mutations are introduced in the r R N A [2,3]. The
second important component of the translation
initiation complex is the start codon, and its spatial relationship to the RBS. A U G is the usual
translation initiation codon in all species investigated so far, although an increasing number of
examples are breaking this general rule. In eucaryotic cells several n o n - A U G triplets are competent for translation initiation [4]. Greater degeneracy in initiation codon recognition is seen in
bacteria since, in Escherichia coli, the codons
G U G and U U G are known to constitute efficient
initiators [5-8], and even other n o n - A U G codons
can be utilized to some extent [9-14]. In this
communication we present evidence indicating
that AUC, A U A and A U U triplets are capable to
initiate translation in E. coli for the case of a cell
326
wall lytic enzyme (HBL). In addition, the triplet
A U C was also competent to initiate the E. coli
/3-galactosidase translation. These observations
contribute to a better understanding of the influence of the third position of the start codon in
the translation initiation.
a translation fusion vector containing the entire
lac operon but missing the promoter, operator,
and translation initiation site as well as the first
six nonessential codons of the lacZ gene coding
for 13-galactosidase [17].
3.2. Site-directed mutagenesis
Point mutations on p H L l l changing the ATC
triplet of the hbl gene to ATA, ATT, and ATG
(see Fig. 1A) were generated on appropriate M13
subclones by phosphorothioate-based oligonucleotide mutagenesis [16] using a kit from Amer-
3. MATERIALS A N D M E T H O D S
3.1. Bacterial strains and plasmids
E. coli DH1 [15], E. coli TG1 [16], and E. coli
MCl116 [17] were used in this study, pSKS107 is
A
Met Asp Arg Asn Arg Leu Arg Thr
TCTA~TCCCCATC GAT AGA AAC AGACTA CGT ACA
H B - 3 DNA EcoRV
f r a g m e n t (1.7 kb)
ligation
+ Sma I
+
"
BAP
A
G
C
~
"
Sst I
T
B
i~i!; iii
/!
G
A
T
A
G
C
T
A
C
C
C
C
T
A
G
h~;~i ¸
/?
~? ?
j
Fig. 1. Construction and sequence of pHLll. (A) Thin lines and letters correspond to pUC13 and thick lines and letters to phage
HB-3 DNA. Upper line in the sequence is the RBS element, (B) Nucleotide sequence of the relevant junction zone. Ap, ampicillin;
BAP, bacterial alkaline phosphatase.
327
sham. All the constructions were checked by the
dideoxy chain-termination sequence analysis [18].'
3.3. Immunoblot analysis
Purified protein and crude extracts were electrophoresed in 10% SDS-polyacrylamide gels and
transferred to nitrocellulose membranes [19]. After blocking the binding sites with reconstituted
dried skim milk, the membrane was subsequently
incubated at room temperature with anti-pneumococcal amidase serum [20], peroxidase-conjugated AffiniPure goat anti-rabbit serum (Jackson Immunoresearch), and 4-chloro-l-naphtol
(Sigma), essentially as recommended by the suppliers.
3.4. Protein purification and amino acid sequence
analysis
Complete and truncated forms of HBL were
purified by affinity chromatography on DEAEcellulose as previously described [21,22]. The Nterminal amino acid sequence of the purified
protein was determined by Edman degradation in
a pulse-liquid phase protein sequencer model
477A (Applied Biosystems).
3.5. Analytical methods and materials
The standard assay conditions for the lytic
activity of HBL on pneumococcal cell walls have
been described previously [23]. /3-Galactosidase
activities were determined essentially as described [24]. Protein concentrations were determined as described [25] and SDS-PAGE by the
method of Laemmli [26].
4. RESULTS AND DISCUSSION
We have recently cloned and expressed in E.
coli the hbl gene from the Streptococcus pneumoniae temperate bacteriophage HB-3 encoding an
N-acetylmuramoyl-L-alanine amidase (HBL) [22].
In the course of subcloning experiments to sequence the hbl gene [27], pilL11 was constructed
(Fig. 1A). This plasmid contains a truncated hbl
gene (Ahbl) lacking the first two codons of the
corresponding open reading frame, i.e., the ATG
initiation codon and the GAT one [27]. Surpris-
- -
43.0
- -
31.0
Fig. 2. Immunoblot analysis of E. coli crude extracts. 1, DH1
(pUC13); 2, DH1 (pHLll-ATC); 3, DH! (pHLll-ATT); 4,
DH1 (pHLll-ATA); 5, DH1 (pHLll-ATG); 6, purified HBL.
Numbers on the right show molecular size in kilodaltons.
ingly, crude sonicated extracts obtained from E.
coli DH1 ( p H L l l ) showed enzymatic activity
when tested on choline-containing pneumococcal
cell wall as a substrate. The enzyme responsible
for this lytic activity shared the relevant biochemical and immunological properties of the phage
(HBL) and pneumococcal (LYTA) amidases [22].
Furthermore, a single band with the same mobility as that of the complete HBL amidase appeared in Western blot analysis (Fig. 2). On the
other hand, a careful inspection of the nucleotide
sequences of the pUC13 vector located upstream
of the hbl gene insert showed no evidence of the
presence of in-frame ATG or GTG triplets that
could serve as initiation signals for translation.
However, a putative RBS sequence [28] (5'AGAGGA-3') is located 5 nucleotides upstream
of an ATC triplet (the third codon of the hbl
gene) (Fig. 1A) suggesting the possibility that this
triplet could behave as a translation initiator. To
test this hypothesis we purified to electrophoretic
homogeneity the lytic AHBL enzyme from E. coli
DH1 ( p H L l l ) and the N-terminal amino acid
sequence analysis of the pure protein (Met-AspArg-Asn-Arg-Leu-Arg-Thr-Gly-Leu-Pro) fully
confirmed the above assumption. This N-terminal
sequence of HBL is the one predicted from the
nucleotide sequence provided that the AUC
codon was translated as N-formyl-methionine and
that this residue was not cleaved off post-translationally, a result that is in agreement with the
data available about the extent of N-terminal
methionine excision from E. coli proteins [29].
328
Since initiation at a n o n - A U G triplet requires
base mismatch at the level of the codon-anticodon interaction, the lytic activity found using
A U C codon as initiator must imply a certain
destabilization at the third position of this interaction. To better study the influence of the third
nucleotide in the initiation codon we changed, by
site-directed mutagenesis, the A U C start codon
to AUA, A U U and AUG. The latter construction
Acc[
TCTAGAGGATCCCC ATC GAC CTG gAG CCA AGC
/
,o,,
~
would measure the maximum level of expression
for the truncated H B L enzyme and should represent a positive control. The results shown in
Table 1 demonstrate that AUC, A U A and A U U
triplets behaved in fact as initiator codons, but
denoted a 20-fold decrease for A U C and A U U
and a 13-fold decrease for A U A in expression
levels when compared to A U G .
To test whether the peculiar response of non-
stl
+Cla[
\
/
A
C
G
T
B
Fig. 3. Construction and sequence of pATCG1. (A) Thin lines and letters correspond to pUC13, thick lines and letters to lac
operon with the fl-galactosidase (Z), permease (Y) and transacetylase (A) genes and white arrows represent the hbl gene. Upper
line in the sequence is the RBS element. (B) Nucleotide sequence of the relevant junction zone.
329
Table 1
Effects of start codon mutations on the expression of Ahbl in
Escherichia colt strain DH1
Start codon
H B L activity a
( U / r a g of protein)
Efficiency b
(%)
AUG
AUC
AUA
AUU
Control c
30 550
1 650
2 290
1 375
< 10
100
5.4
7.5
4.5
0
H B L assays were done with sonicated crude extracts at 37°C
for 10 rain, with preincubation at 4°C for 5 rain, as previously described I23].
b Efficiencies are expressed as a ratio of H B L activity relative
to the value obtained with A U G as initiation codon.
c Crude sonicated extracts from E. colt DH1 (pUC13).
AUG triplets to initiate the translation machinery
was not restricted to the particular case of the hbl
gene described above, we extended our observations to a well-known gene. For this purpose, we
took advantage of the /3-galactosidase structural
gene lacZ from pSKS107 by inserting it into
pilL11 in an identical position to that of hbl, and
with an in-frame ATC codon as putative initiator.
The recombinant plasmid pATCG1 is, as in the
case of pilL11, a pUC-derivative containing identical upstream sequences and the presumed ATC
codon as initiator of the lacZ gene (Fig. 3A). The
accuracy of the construction of pATCG1 was
verified by sequencing the junction zone (Fig. 3B)
and the plasmid was used to transform competent
cells of E. colt MCl116. The appearance of blue
colonies on 5-bromo-4-chloro-3-indolyl /3-Dgalactopyranoside (X-gaD-containing plates suggested the expression of sufficient amounts of
/3-galactosidase to hydrolyze the chromogenic
substrate. This was confirmed by measuring the
enzymatic activity of crude sonicated extracts from
E. colt M C l l l 6 (pATCG1). The /3-galactosidase
activity in these ceils (300 U / m g total protein)
indicated a moderate but significant level of expression provided that E. colt M C l l l 6 crude
sonicated extracts did not produce detectable levels (less than 2 U/rag total protein).
The above results provide evidence on the
ability of the AUC, AUA and AUU triplets to
serve as initiation translation signal for the pneu-
mococcal cell wall lytic enzyme (HBL) and, at
least, of the AUC triplet for initiation of the
/3-galactosidase. Whether this ability is restricted
to polypeptide-encoding sequences showing as
starting codons AUC, AUA or AUU preceded by
the same elements present in p H L l l and
pATCG1, or the presence of those codons within
a variety of transcription and translation signals
could initiate protein translation remains to be
investigated.
The moderate efficiency of initiation of protein synthesis for non-AUG triplets found in this
work would recall Crick's wobble hypothesis,
which suggested that the pairing between codon
and anticodon at the first two codon positions
always follows the usual rules, but exceptional
'wobbles' might occur at the third position [30].
These unconventional base pairings occur due to
the conformation of the tRNA anticodon loop,
that allows unusual flexibility at the first base of
the anticodon. In our particular case the available
data on destabilizing effects of specific single
base mismatches in RNA seems insufficient to
conclude whether the strength of the codon-anticodon interaction is the only determinant
responsible for the moderate initiation efficiencies of the non-AUG triplets.
Several non-AUG triplets capable to initiate
the protein synthesis in procaryotes are known
[7,9,11]. In fact, seven out of the nine possible
initiator triplets with a single nucleotide variant
with respect to the universal initiator AUG have
been reported as start codons. However, a remarkable discrepancy between the data on the
translation efficiency of these non-AUG codons
has been found, since in these reports the gene
elements vary in every case and no general rule
has been drawn about this subject [7,9,10,12]. In
contrast, in the HBL protein studied here, the
relative translation efficiencies for the start
codons were fully comparable (Table 1) since
only the third position of the initiator triplets
were changed, and, in turn, this would be the
unique reason responsible for such variation in
translation efficiencies.
With the exception of ACG, the start codons
capable of initiating contain two contiguous bases
complementary to the fmet tRNA anticodon. Our
330
results c o n f i r m this ambiguity in the c o d o n - a n t i c o d o n i n t e r a c t i o n , referred to the third position
of the c o d o n a n d m a i n t a i n i n g the same o r d e r of
t r a n s c r i p t i o n a l a n d t r a n s l a t i o n a l elements. In this
sense, we can c o n c l u d e that A U C , A U A a n d
A U U triplets f u n c t i o n as i n i t i a t i o n codons at a
similar m o d e r a t e efficiency, a p e c u l i a r p r o p e r t y
suggesting that, in c e r t a i n a r r a n g e m e n t s of gene
elements, n a t u r e can exploit this unspecificity in
start t r a n s l a t i o n to regulate i m p o r t a n t f u n c t i o n s
[31] or to build new p o l y p e p t i d e s that could have
accomplished some role in p r o t e i n evolution.
ACKNOWLEDGEMENTS
W e t h a n k R. L6pez, E. Garcfa a n d J.L. Garcfa
for useful discussions a n d for the critical r e a d i n g
of the m a n u s c r i p t . This work was s u p p o r t e d by a
g r a n t from D i r e c c i 6 n G e n e r a l de Investigaci6n
Cientifica y T 6 c n i c a (PB87-0214). A.R. was the
r e c i p i e n t of a fellowship from Plan de F o r m a c i 6 n
del P e r s o n a l Investigador.
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