Molecular Microbiology (1998) 27(5), 987–1001
An AUG initiation codon, not codon–anticodon
complementarity, is required for the translation of
unleadered mRNA in Escherichia coli
William J. Van Etten and Gary R. Janssen*
Department of Microbiology, Miami University, Oxford,
OH 45056, USA.
Introduction
The translational efficiency, or protein yield per unit message, of a specific coding sequence (CDS) is limited by
ribosome binding and is a function of the information presented by mRNA elements within the ribosome binding
site (RBS) and their recognition by initiating ribosomes.
The RBS of conventionally leadered Escherichia coli
mRNA extends approximately 6 15 nucleotides relative
to the start codon (Steitz, 1969; Steitz and Jakes, 1975;
Steitz and Steege, 1977), contains non-random sequence
using statistical analysis (Scherer et al ., 1980; Stormo et
al ., 1982), and is responsible for establishing a 1000-fold
range of translational efficiencies (Ray and Pearson, 1974;
1975). Highly efficient initiation regions include some or
all of the following mRNA elements: a polypyrimidine
tract for ribosomal protein S1 interaction (Boni et al .,
1990; Zhang and Deutscher, 1991; Tzareva et al ., 1994);
a Shine–Dalgarno (SD) sequence with basepairing complementarity to the anti-SD (ASD) of the 16S rRNA
(Shine and Dalgarno, 1974; Hui and de Boer, 1987;
Jacob et al ., 1987); a cognate start codon for initiator
fMet-tRNA anticodon interaction (Ringquist et al ., 1992;
Vollenoweth and Rabinowitz, 1992); and base-specific
enhancer sequences upstream (Olins and Rangwala,
1989) or downstream (Sprengart et al., 1990; 1996) of the
start codon. The relative importance and interdependence
of these mRNA elements as well as the temporal order of
their recognition by initiating ribosomes is less well understood. Whether the 30S subunit, along with initiation factors, binds first to mRNA and then to fMet-tRNA or vice
versa is not known. In vitro evidence suggests that the
30S subunit binds to mRNA or to fMet-tRNA in a random
order and in rapid equilibrium to form a 30S-mRNA–
fMet-tRNA ternary complex, followed by a rate-limiting
‘rearrangement’ to form the 30S ‘initiation complex’ (Gualerzi and Pon, 1990). However, in vivo evidence suggests
that, at least for some mRNAs, the 30S subunit binds first
to the fMet-tRNA, delivered by IF2, and then binds the
mRNA (Wu et al ., 1996; Wu and RajBhandary, 1997).
The SD sequence, within the untranslated leader, provides
for an increase in the Ka of 30S-mRNA binary complex formation, but does not influence mechanistically the formation of the ‘initiation complex’, the rate of initiation, or the
establishment of reading frame (Gualerzi and Pon, 1990).
Summary
We determined the in vivo translational efficiency of
‘unleadered’ lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD)
sequence in Escherichia coli and found that changing
the SD sequence of leadered lacZ from the consensus
58-AGGA-38 to 58-UUUU-38 results in a 15-fold reduction in translational efficiency; however, removing
the leader altogether results in only a twofold reduction. An increase in translation coincident with the
removal of the leader lacking a SD sequence suggests
the existence of stronger or novel translational signals
within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation
codon to other naturally occurring initiation codons
(GUG, UUG, CUG) in the presence and absence of a
leader and find that mRNAs lacking leader sequences
are dependent upon an AUG initiation codon, whereas
leadered mRNAs are not. This suggests that mRNAs
lacking leader sequences are either more dependent
on perfect codon–anticodon complementarity or
require an AUG initiation codon in a sequence-specific
manner to form productive initiation complexes. A
mutant initiator tRNA with compensating anticodon
mutations restored expression of leadered, but not
unleadered, mRNAs with UAG start codons, indicating
that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader
sequences. These data suggest that a cognate AUG
initiation codon specifically serves as a stronger and
different translational signal in the absence of an
untranslated leader.
Received 29 August 1997; revised 19 November 1997; accepted 8
December 1997. *For correspondence. E-mail grjanssen@miavx1.
muohio.edu; Tel. (513) 529-5448; Fax (513) 529-2431
Q 1998 Blackwell Science Ltd
m
988 W. J. Van Etten and G. R. Janssen
Naturally leaderless messages are devoid of the untranslated leader, the SD sequence, and any other translational
signals contained therein. Other than the probable importance of the initiation codon, the translational signals within
leaderless mRNA are largely unknown. There is evidence
to suggest that a 58 terminal AUG initiation codon may be
sufficient to establish the start site and reading frame for
leaderless mRNA (Jones et al ., 1992). Four different initiation codons are known to occur naturally in leadered mRNA
of E . coli : AUG (<90%), GUG (<8%), UUG (<1%) and
AUU (very rarely) (Schneider et al ., 1986). In the presence
of an untranslated leader, non-cognate start codons support translation, albeit somewhat less well (Ringquist et
al ., 1992). Two actinomycete leaderless mRNAs initiate
with a GUG start codon (ermE , Bibb et al ., 1994; malR ,
van Wezel et al ., 1997); however, their expression relative
to leaderless mRNAs initiating with AUG start codons is
unknown.
Genes that encode naturally leaderless mRNA are relatively infrequent, although more than 30 have been identified (summarized in Wu and Janssen, 1996) since the E .
coli phage l c I repressor was first reported in 1976
(Ptashne et al., 1976). Observations of leaderless mRNA
(Janssen, 1993) in Bacteria, Archaea, Eukarya and eukaryotic organelles suggest that sequence and/or structural
information contained within the CDS are sufficient to signal the translational start site and reading frame in these
diverse biological systems. The widespread occurrence
of leaderless mRNA suggests that translation of leaderless mRNA might represent a fundamental capability of
all translation systems. In possible support of this, it has
also been shown that leaderless mRNAs are translated
in heterologous hosts (Janssen, 1993; Wu and Janssen,
1997).
In addition to naturally leaderless mRNA, the untranslated leader may be removed from a conventionally leadered message without the loss of translatability (Wu
and Janssen, 1996), suggesting that the initiation codon
and downstream CDS are sufficient to signal translation.
After removal of the untranslated leader, it is not known
whether the remaining mRNA elements within the CDS
serve the same function, or take on some novel function
contributing to translation initiation. Presumably, the initiation codon retains at least similar function in the presence
or absence of a leader by providing base-pairing complementarity to the anticodon of the initiator tRNA. Observations that mRNA is translated after the removal of its
untranslated leader (Wu and Janssen, 1996) provide an
opportunity to characterize features within the CDS that
determine the translational efficiency of mRNA independent of elements within the leader. In this study, we examine the role of the initiation codon in translation of mRNA
lacking an untranslated leader sequence.
Results
Construction of leadered and unleadered lacZ with
alternate start codons
To investigate the role of the initiation codon in translation,
both in the presence and in the absence of an untranslated
leader, seven site-directed mutations were constructed
from plasmids containing a lacZ reporter gene with (pSDAUG), or without (pUL-AUG), an untranslated leader
(G. R. Janssen, unpublished) (Fig. 1). Four mutations
were constructed from pSD-AUG that alter the consensus
SD sequence from 58-AGGA-38 to 58-UUUU-38 (pSD-KO)
or the lacZ initiation codon from AUG to GUG, UUG, and
CUG (pSD-GUG, pSD-UUG, and pSD-CUG, respectively). Similarly, three mutants were constructed from
pUL-AUG to change the lacZ initiation codon from AUG
Fig. 1. Physical map of leadered and unleadered lacZ expression
vectors with alternate start codons.
DNA sequences above the plasmid map report the differences
between wild-type lacZ (first), leadered lacZ with 58-UUUU-38
substitution of the SD sequence (second; pSD-KO), leadered lacZ
(third; pSD-NUG) and unleadered lacZ (fourth; pUL-NUG) with
alternate start codons. The Nco I site (58-CCATGG-38) is lost when
A is C, G, or T. Abbreviations and indications: sequences
identifying the lac promoter ¹10 region and the SD sequence are
overlined; lowercase indicates an untranslated leader; asterisk
indicates transcriptional start site; N indicates A, G, T or C; bla ,
b-lactamase gene; ori , pBR322 origin of replication; rrnB t, E . coli
rrnB T1 and T2 transcriptional terminators; T1, E . coli rrnB T1
transcriptional terminator; lacZ , E . coli b-galactosidase gene.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 989
to GUG, UUG, and CUG (pUL-GUG, pUL-UUG and pULCUG respectively).
Removal of a leader with a poor SD sequence
restores expression but results in dependence on
AUG as the initiation codon
b-Galactosidase assays were performed on mid-log
phase cultures of the nine constructs containing leadered
and unleadered lacZ with alternate start codons (Fig. 2A).
Changing the SD sequence from 58-AGGA-38 to 58-UUUU38 when comparing pSD-AUG with pSD-KO results in a 20fold reduction of lacZ expression; however, removing the
leader altogether when comparing pSD-AUG to pUL-AUG
results in only a twofold reduction. Altering the start
codon of leadered lacZ from AUG to GUG, UUG or CUG
reduces expression linearly over a 10-fold range. However, in the absence of an untranslated leader, altering
the lacZ start codon results in expression near background
levels. The lacZ expression from pUL-GUG and pUL-UUG
is marginally distinguishable from the host negative control
by enzyme assay. In addition, they have a faint blue colour
upon extended incubation on LB agar with X-gal, also indicating a low level of expression. The lacZ expression from
pUL-CUG is not detectable by enzyme assay or by
extended incubation on LB agar with X-gal.
Transcriptional start site is not altered by the change
in the initiation codon
The transcriptional start sites of unleadered lacZ with
alternate start codons were identified by primer extension
(Fig. 3). Transcription of unleadered lacZ with a cognate
start codon initiates at the A residue of the AUG start
codon and is not changed in position by single base substitution at the þ1 position from A to G, U or C. The leadered
lacZ constructs with alternate start codons initiate transcription at an A residue at the 58 end of a 38 nucleotide
untranslated leader. The transcriptional start site is unaffected by the single base substitutions at the þ1 position
of the translational start site (Fig. 4).
Steady-state message levels are not affected by the
removal of the untranslated leader or change in the
start codon
Steady-state message levels of leadered and unleadered
lacZ with alternate start codons are shown in Fig. 5
using Northern dot-blot analysis. Total RNA from the
nine constructs and the host negative control were probed
with an end-labelled oligonucleotide specific for lacZ (Fig.
5A). After scanning for beta-emission and autoradiography, the blot was stripped and reprobed with an endlabelled oligonucleotide specific for the 16S rRNA as an
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
Fig. 2. b-Galactosidase activity, steady-state lacZ mRNA level, and
translational efficiency of leadered and unleadered lacZ with
alternate start codons.
A. b-Galactosidase activity of leadered and unleadered lacZ with
alternate start codons and the host negative control. A value of
99 Miller units is represented by 100%.
B. Steady-state lacZ mRNA levels by Northern dot-blot analysis
after normalizing to the 16S rRNA internal control.
C. Translational efficiency; a ratio of b-galactosidase activity and
steady-state lacZ mRNA. b-Galactosidase activity, lacZ mRNA
levels, and translational efficiency are expressed relative to the
values measured for pUL-AUG (100%). Error bars represent the
standard deviation.
990 W. J. Van Etten and G. R. Janssen
Fig. 3. Transcriptional start sites of
unleadered lacZ with alternate start codons
by primer extension analysis. The DNA
sequence indicated is the sense strand from
¹75 (top) to þ14 (bottom) relative to the
translational start site (þ1); the start codon
and the promoter ¹35 and ¹10 regions are
underlined; N indicates A, G, T, or C. Lanes
G, A, T and C indicate the dideoxy
termination sequencing reactions.
Transcriptional start sites are indicated on the
DNA sequence with an asterisk. Lanes H, PA ,
PG, PU and PC represent primer extension
reaction products resulting from RNA isolated
from E . coli RFS859 (H), and E . coli RFS859
containing pUL-AUG (PA ), pUL-GUG (PG ),
pUL-UUG (PU ) or pUL-CUG (PC ).
internal control (Fig. 5B). The steady-state lacZ mRNA
levels are not significantly affected by the change in the
SD sequence, the removal of the untranslated leader or
the change in the initiation codon (Fig. 2B).
An AUG start codon is required for efficient
translation of unleadered, but not leadered, lacZ
The translational efficiency (Fig. 2C), or protein yield per
unit message, was determined for each of the constructs
using b-galactosidase activity as a measure of the LacZ
protein yield and Northern hybridization as a measure of
the lacZ steady-state mRNA levels. Translational efficiencies closely resemble b-galactosidase activities because
of the relatively consistent mRNA levels among the constructs. Altering the SD sequence from 58-AGGA-38 to
58-UUUU-38 results in a 15-fold reduction in translational
efficiency when comparing pSD-AUG with pSD-KO. However, removal of the leader altogether restores translational
efficiency to nearly half the efficiency of leadered lacZ with
the consensus SD sequence when comparing pUL-AUG
with pSD-AUG. In the presence of a leader and SD
sequence, initiation at AUG is more efficient than GUG,
greater than twice that of UUG, and nearly eightfold
greater than CUG. However, within statistical standard
error, unleadered lacZ with the non-cognate start codons
do not support translation above background levels.
The requirement for an AUG start codon in the
absence of an untranslated leader is independent of
the coding sequence
To determine if the requirement for an AUG start codon in
the absence of an untranslated leader is a general feature
of an unleadered mRNA or an effect specific to lacZ ,
leadered and unleadered gusA with GUG, UUG, and
CUG start codons were constructed (pSD-GUGgus, pSDUUGgus, pSD-CUGgus, pUL-GUGgus, pUL-UUGgus,
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 991
Fig. 4. Transcriptional start sites of leadered
lacZ with alternate start codons by primer
extension analysis.
The DNA sequence indicated is the sense
strand from ¹113 (top) to þ14 (bottom)
relative to the translational start site (þ1); the
start codon and the promoter ¹35 and ¹10
regions are underlined; N indicates A, G, T,
or C. Lanes G, A, T and C indicate the
dideoxy termination sequencing reactions.
Transcriptional start sites are indicated on the
DNA sequence with an asterisk. Lanes PA ,
PG, PU and PC represent primer extension
reaction products resulting from RNA isolated
from E . coli RFS859 containing pSD-AUG
(PA ), pSD-GUG (PG ), pSD-UUG (PU ) or
pSD-CUG.
and pUL-CUGgus respectively). As a negative control, the
¹10 region of the lac promoter in pUL-AUGgus was changed from TATAAT to TATAAC by cloning a mutant, nonfunctional Plac* promoter fragment (Wu and Janssen,
1996) into pUL-AUGgus to make pUL-10mtgus. b-Glucuronidase assays were performed on mid-log phase cultures of the nine constructs and the host control (Fig. 6).
Similar to what was observed with the lacZ CDS (Fig.
2A), the untranslated leader provides a fivefold stimulation
of gusA expression when comparing pSD-AUGgus with
pUL-AUGgus. Altering the start codon of leadered gusA
from AUG to GUG, UUG, or CUG reduces expression in
a manner similar to that of leadered lacZ (Fig. 2A) with
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
the alternate start codons. Altering the gusA start codon
in the absence of an untranslated leader resulted in
expression near background levels. Primer extension analysis confirmed the predicted transcriptional start sites of
leadered and unleadered gusA with the alternate start
codons (Fig. 7). Transcription initiates at the þ1 and þ3
positions from pUL-UUGgus and the þ1 and þ2 positions
from pUL-CUGgus; however, the absence of gusA enzyme
activity from E . coli containing these plasmids (Fig. 6) indicates the mRNA initiating at þ1 was not translated. The
simultaneous loss of transcription and b-glucuronidase
activity associated with the lac promoter mutation (when
comparing pUL-AUGgus with pUL-10mtgus) confirmed
992 W. J. Van Etten and G. R. Janssen
Fig. 5. Steady-state message levels of
leadered and unleadered lacZ with alternate
start codons by Northern dot-blot analysis.
A and B are autoradiograms of the same
Northern dot blots probed with lacZ and 16S
rRNA specific end-labelled oligonucleotides
respectively. Labels on the left indicate the
source of RNA bound to the membrane
including the RFS859 host strain and tRNA
negative controls. Samples 1 and 2 identify
separate RNA preparations and labels on the
bottom indicate the mass of total cellular RNA
bound to the membrane. The upper two rows,
showing Northern hybridization of RNA from
pUL-AUG and pSD-KO, are from an
independent blot which also contained the
additional control RNA from pSD-AUG and
the host strain, but were not shown for clarity.
that the observed expression from pUL-AUGgus resulted
from translation of the unleadered mRNA.
The specific requirement for an AUG start codon in
the absence of an untranslated leader is not to
provide for codon–anticodon complementarity
To determine if the requirement for an AUG start codon in
the absence of an untranslated leader is to provide for
codon–anticodon complementarity, plasmids containing
leadered and unleadered lacZ with AUG and UAG start
codons (pACSD-AUG, pACSD-UAG, pACUL-AUG,
pACUL-UAG, pACUL-UUAG) were constructed and transformed into a lacZ deletion strain (E . coli RFS859) harbouring a compatible high-copy-number plasmid with
(pAmSUPþ), or without (pAmSUP¹), an amber-suppressing initiator tRNA (Wu et al ., 1996).
b-Galactosidase assays were performed on the five
constructs in the presence [þ], or absence [¹], of the suppressor tRNA (Fig. 8A). Similar to previous observations
(Figs 2A and 6), the untranslated leader provides an
approximate twofold stimulation of expression when translation initiates at an AUG start codon in the absence of the
suppressor tRNA (pACSD-AUG[¹] vs. pACUL-AUG[¹]).
As expected, there is no expression from leadered
(pACSD-UAG[¹]) or unleadered (pACUL-UAG[¹] and
pACUL-UUAG[¹])lacZ with UAG start codons in the
absence of the suppressor tRNA. The suppressor tRNA
restores expression from leadered lacZ with a UAG
start codon (pACSD-UAG[þ]), but not unleadered lacZ
with a UAG start codon (pACUL-UAG[þ]and pACULUUAG[þ]).These results indicate that an initiator tRNA
with compensating anticodon mutations is sufficient for
the suppression of a UAG initiation codon on a leadered
lacZ ; however, codon–anticodon complementarity is
insufficient for translation of unleadered lacZ . The transcriptional start sites of pACSD-UAG, pACUL-UAG, and
pACUL-UUAG were identified by primer extension analysis (Fig. 9). Transcription of leadered lacZ with a UAG
start codon initiates at the predicted A residue at the 58
Fig. 6. b-Glucuronidase activity of E . coli DH5a containing
leadered and unleadered gusA with alternate start codons. Error
bars indicate the standard deviation. A value of 471 units is
represented by 100%.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 993
Fig. 7. Transcriptional start sites of leadered
and unleadered gusA with alternate start
codons by primer extension analysis. The
DNA sequence indicated is the sense strand
of pSD-AUGgus (left) from ¹113 (top) to þ14
(bottom) and pUL-AUGgus (right) from ¹75
(top) to þ14 (bottom) relative to the
translational start site (þ1); the start codon
and the promoter ¹35 and ¹10 regions are
underlined; N indicates A, G, T, or C.
Lanes G, A, T and C indicate the dideoxy
termination sequencing reactions.
Transcriptional start sites are indicated on the
DNA sequences with an asterisk. Lanes H,
PSA , PSG, PSU, PSC, PUA , PUG, PUU, PUC and
P¹10 represent primer extension reaction
products resulting from RNA isolated from
E . coli DH5a (H), and E . coli DH5a
containing pSD-AUGgus (PSA ), pSD-GUGgus
(PSG ), pSD-UUGgus (PSU ), pSD-CUGgus
(PSC ), pUL-AUGgus (PUA ), pUL-GUGgus
(PUG ), pUL-UUGgus (PUU ), pUL-CUGgus
(PUC ) or pUL-10mtgus (P¹10 ).
end of the 38 nucleotide leader; however, transcription of
unleadered lacZ with a UAG start codon initiates at both
the U and A residues of the UAG start codon. A greater
fraction of the transcription signal is observed at the A residue of the UAG start codon which was the justification for
constructing pACUL–UUAG. Transcription of lacZ with a
UAG start codon and a single nucleotide leader (UUAG)
initiates entirely at the first U residue at the ¹1 position
of the potential translational start site.
Initiator codon–anticodon complementarity alone is
insufficient for the translation of naturally leaderless
mRNA
As leaderless mRNA with GUG initiation codons occur in
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
nature (ermE , Bibb et al ., 1994; malR , van Wezel et al .,
1997), we suspected that naturally leaderless mRNA
might have additional or stronger translational signals
within the CDS not present within unleadered mRNAs
that might confer a lesser dependence on an AUG initiation codon. In test of this possibility, we fused the first 16
codons of the naturally leaderless l c I to a lacZ reporter
and constructed plasmids containing leadered and leaderless c I– lacZ fusions with AUG and UAG start codons
(pSD-AUGcI, pSD-UAGcI, pUL-AUGcI, pUL-UAGcI,
pUL-UAUAGcI). The plasmids were transformed into a
lacZ deletion strain (E . coli RFS859) harbouring a compatible high-copy-number plasmid with (pAmSUPþ), or
without (pAmSUP¹), an amber-suppressing initiator
tRNA (Wu et al ., 1996) to determine if codon–anticodon
994 W. J. Van Etten and G. R. Janssen
Fig. 8. b-Galactosidase activity of leadered and unleadered lacZ or
c I-lacZ fusions with AUG or UAG start codons in the presence and
absence of an amber-suppressing initiator f-Met tRNA.
A. b-Galactosidase activity of E . coli RFS859 containing the
leadered or unleadered lacZ constructs with AUG or UAG start
codons in the presence [þ] or absence [¹] of a plasmid containing
the amber-suppressing initiator tRNA. A value of 213 Miller units is
represented by 100%.
B. b-Galactosidase activity of E . coli RFS859 containing the
leadered or unleadered c I-lacZ fusion constructs with AUG or UAG
start codons in the presence [þ] or absence [¹] of the ambersuppressing initiator tRNA. A value of 11 025 Miller units is
represented by 100%. Error bars represent the standard deviation.
complementarity alone was sufficient for the translation of
a naturally leaderless mRNA.
b-Galactosidase assays were performed on the five
constructs in the presence [þ] or absence [¹] of the
suppressor tRNA (Fig. 8B). Similar to previous observations (Figs 2A, 6 and 8A), the untranslated leader provides
an approximate twofold stimulation of expression when
translation initiates at an AUG start codon in the presence
or absence of the suppressor tRNA (pSD-AUGcI[þ]/[¹]
vs. pUL-AUGcI[þ]/[¹]). However, the stimulation of c IlacZ expression might be a falsely low indication of the difference in translatability because leadered expression is
approaching the upper limit we have observed in this
host strain (J. Martin-Farmer, A. Walker, G. Janssen,
unpublished). As expected, there is no expression from
leadered (pSD-UAGcI[¹]) or leaderless (pUL-UAGcI[¹]
and pUL-UAUAGcI[¹]) c I– lacZ with UAG start codons in
the absence of the suppressor tRNA. As was observed
with lacZ (Fig. 8A), the suppressor tRNA restores expression from the leadered c I– lacZ with a UAG start codon
(pSD-UAGcI[þ]), but there is relatively little suppression
of the c I– lacZ UAG initiation codon in the absence of a leader (pUL-UAGcI[þ] and pUL-UAUAGcI[þ]). Although
expression from pUL-UAGcI[þ] and pUL-UAUAGcI[þ]
are only one to two percentage of that observed from
pUL-AUGcI[þ]/[¹], the level of lacZ expression (100 and
200 Miller units respectively) indicates that the requirement for an AUG initiation codon in the absence of a leader
is not absolute, however, far more stringent than in the
presence of a leader.
The transcriptional start sites of pSD-UAGcI, pULUAGcI, and pUL-UAUAGcI were identified by primer extension analysis (W. Van Etten, unpublished). Transcription of
leadered c I– lacZ with an AUG or UAG start codon initiates
at the predicted A residue at the 58 end of the 38 nucleotide
leader. Transcription of the leaderless c I– lacZ fusion initiates at the A of the c I AUG start codon. However, as was
observed with pACUL-UAG (Fig. 9), transcription of leaderless c I– lacZ with a UAG start codon initiates at both
the U and A residues of the UAG start codon which was
the justification for constructing pUL-UAUAGcI. Transcription of the two nucleotide leadered c I– lacZ with a UAG
start codon (pUL-UAUAGcI) initiates entirely at the first
U residue at the ¹2 position of the translational start site.
Discussion
mRNA elements within the CDS serve as greater
and/or different signals to translation in the absence
of an untranslated leader
The degree of complementarity between the SD sequence
within the 58 untranslated leader of prokaryotic mRNA and
the ASD sequence near the 38 end of the 16S rRNA is a
major determinant of the efficiency by which leadered
mRNAs are translated (Hui and de Boer, 1987; Jacob et
al., 1987; Ringquist et al ., 1992). Some naturally leaderless
messages, although lacking a conventional SD–ASD interaction, are highly expressed [e.g. aph (Janssen et al .,
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 995
Fig. 9. Transcriptional start sites of leadered
and unleadered lacZ with amber start codons
by primer extension analysis. The DNA
sequencing reactions are pACSD-UAG (left),
pACUL-UAG (middle) and pACUL-UUAG
(right). The DNA sequence indicated is the
sense strand of pACSD-UAG (left) from ¹113
(top) to þ14 (bottom) and pACUL-UUAG
(right) from ¹75 (top) to þ14 (bottom) relative
to the translational start site (þ1); the start
codon and the promoter ¹35 and ¹10
regions are underlined. Lanes G, A, T, and C
indicate the dideoxy termination sequencing
reactions. Transcriptional start sites are
indicated on the DNA sequences with an
asterisk. Lanes PSAm, PUAm and PUUAm
represent primer extension reaction products
resulting from RNA isolated from E . coli
RFS859 containing pACSD-UAG[þ](PSAm ),
pACUL-UAG[þ](PUAm ), and pACULUUAG[þ](PUUAm ).
1989), bop (Stoekenius et al ., 1979), rph (Hoshiko et al .,
1988)]; however, little is known of the mRNA features that
determine their translational efficiency. If these highly
expressed leaderless mRNAs are also translated efficiently, what serves as the translational signal in the
absence of the SD that allows them to effectively compete
with leadered mRNA for initiating ribosomes?
We observe a broad range of expression from the
reporter genes used in this study. Compared with previous
reports (Miller, 1992), lacZ expression is relatively low
and the c I– lacZ is very highly expressed. Based on
band intensity on a Coomassie-stained protein gel, gusA
is expressed at moderate levels (J. Martin-Farmer, personal communication). However, all three reporter genes
show only a modest, relative increase of expression
(lacZ , twofold; gusA , fivefold; c I– lacZ , twofold) in the presence of a modified lac untranslated leader sequence. The
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
translational efficiency of unleadered relative to leadered
mRNA has been determined for only the lacZ reporter;
however, with estimation of the mRNA levels provided
by the intensity of primer extension reaction signals,
the relative translational efficiencies of the c I– lacZ (W.
Van Etten, unpublished) and gusA reporter genes are
likely to be consistent with what has been determined
for lacZ .
In this study, we have determined the relative translational efficiency of a leadered and unleadered lacZ and
find that mutation of the SD sequence from the consensus
to 58-UUUU-38 results in a dramatic loss of translatability,
however, removal of the leader altogether restores translation to nearly half the efficiency of leadered mRNA with a
consensus SD sequence. If the translational signals within
the leader and CDS were additive, one would predict the
translational efficiency of unleadered lacZ to be similar
996 W. J. Van Etten and G. R. Janssen
to, or less than, the translational efficiency of a leadered
lacZ with a poor SD sequence. Our observations suggest
that the translation signals within the leader and CDS are
not necessarily additive and that either novel signals are
created upon the removal of the leader or that signals
within the CDS serve as greater signals in the absence
of the leader.
An AUG initiation codon is required for translation
after removal of the untranslated leader
If the start codon were to serve as a greater signal in the
absence of a leader, one might predict a greater dependence on a cognate initiation codon for the translation of
leaderless and unleadered mRNAs. In the presence of
an untranslated leader, the expression from gusA and
lacZ mRNAs with non-cognate start codons is reduced in
a manner consistent with previous observations (Ringquist
et al ., 1992; Vollenoweth and Rabinowitz, 1992; Sussman
et al ., 1996). However, in the absence of an untranslated
leader there is negligible expression from mRNAs with
non-cognate start codons. These data suggest either an
increased dependence on perfect codon–anticodon complementarity or a sequence-specific requirement for an
AUG start codon for translation after removal of the
untranslated leader sequence.
Codon-anticodon complementarity is insufficient for
translation in the absence of an untranslated leader
If translation had an increased dependence on perfect
codon–anticodon complementarity in the absence of a
leader, one would predict that providing the compensating
mutations to the initiator tRNA anticodon would restore
expression to leaderless mRNA with non-cognate initiation
codons. However, the amber-suppressing initiator tRNA
restores expression to leadered, but not unleadered or leaderless, mRNAs with UAG start codons. This indicates
that an AUG start codon, not codon–anticodon complementarity, is necessary for translation of unleadered and
possibly leaderless mRNA. However, an alternative explanation might be that the fMet-tRNA anticodon can stably
interact with both internal and 58 terminal AUG codons,
whereas the suppressor tRNA anticodon can only interact
with internal UAG codons.
Recognition of the AUG start codon in the absence
of an untranslated leader
If the start codon serves as a stronger and/or different signal for translation in the absence of a leader, how is the
same start codon affected differently after removal of the
leader? The initiator tRNA, IF3, and the ribosome are
the known translation components that recognize and
inspect the initiation codons of leadered mRNA. Messenger RNAs lacking an untranslated leader require more
than codon–anticodon complementarity for expression,
suggesting that either the initiator tRNA, IF3, or the mechanism of translation initiation are different for translation of
mRNAs in the absence of an untranslated leader.
Observations that naturally leaderless mRNAs are found
in a variety of biological systems (Janssen, 1993) and that
most organisms contain a single isoacceptor of initiator
tRNA (RajBhandary and Chow, 1995) make it unlikely
that a novel initiator tRNA is used for the translation of
leaderless messages.
IF3 functions to selectively destabilize initiation complexes such that those containing fMet-tRNA bound to
AUG are favoured (Risuleo et al ., 1976). For leadered
mRNA, the first nucleotide of the initiation codon does
not significantly influence selection by IF3 (Hartz et al .,
1990). Thus, in the presence of a leader, GUG and UUG
initiation codons can be used efficiently as translational
start sites in vivo (Gold, 1988). In addition, CUG has also
been observed to function as an initiation codon (Childs
et al ., 1985). Perhaps IF3 is more discriminating in its
inspection of codon–anticodon interactions at the 58 end
of a mRNA and rejects all non-paired codon–anticodon
interactions. However, if this were true, one would expect
suppression of pUL-UAG in the presence of the ambersuppressing initiator tRNA. Alternatively, IF3 could reject
all non-AUG potential initiation codons when inspecting
at the 58 end of mRNA. However, IF3 does not appear
to discriminate the nucleotide at the first position of the
initiation codon on leadered mRNA in vitro (Hartz et al .,
1990).
A novel mechanism based on additional discrimination
of non-cognate start codons in the absence of a leader
would not explain how existing translational signals within
the CDS serve as greater signals in the absence of a leader. We favour, therefore, a model by which leaderless
mRNAs are recognized for translation by virtue of their
58 terminal AUG specifically prior to, or independent of,
codon–anticodon discrimination by IF3. In support of an
alternative translation mechanism for leaderless mRNA,
it has been demonstrated that leaderless mRNA are capable of forming ternary complexes at the 58 terminal AUG
codon with tightly coupled 70S ribosomes in vitro (Balakin
et al ., 1992). These data might indicate that in vivo translation of leaderless mRNA occurs, in part, after binding
directly to 70S ribosomes without competition from leadered mRNA. Binding of leaderless mRNA to 70S monosomes is not affected by initiation factors and binds as
efficiently as 30S subunits supplemented with initiation
factors (Balakin et al ., 1992). In contrast to 30S subunits,
70S monosomes are unable to form ternary complexes
at internal initiation regions of leadered mRNA (Balakin
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 997
et al ., 1992). Although 70S tight couples are thought to be
a natural and rather abundant form of ribosome in the prokaryotic cell (Noll et al ., 1973), there is presently no direct
evidence that they initiate translation on leaderless
mRNAs in vivo .
Translation in the absence of a leader
The untranslated leader regions from lacZ and gusA are
not necessary for their translation, and addition of a SDcontaining leader sequence to the CDS of lacZ , gusA or
c I– lacZ stimulates expression only two- to fivefold, indicating that leaderless mRNA compete well with leadered
mRNA in vivo . Mutation of the SD sequence away from
consensus results in a dramatic reduction in the translational efficiency of lacZ , however, removing the leader
altogether partially restores translation, indicating the existence of stronger or novel translational signals within the
CDS in the absence of the leader. The initiation codon
serves as a stronger or different translational signal in
the absence of a leader, as indicated by the stringent
dependence on AUG. Why some mRNAs lack untranslated leader sequences is unknown. Work described
here suggests that translation of leaderless mRNA might
initiate by a mechanism different from leadered mRNA.
Analysis of leaderless mRNA might provide additional
insight to the biological and regulatory significance of
untranslated leaders as well as contribute to our general
understanding of mRNA translation signals and the
mechanism of translation initiation.
Experimental procedures
Bacterial strains
E . coli DH5a (F8, endA1, hsdR17, supE44, thi -1, recA1, gyrA ,
relA1, D[lacIZYA -argF ), U 169, deoR , [f80dlac D(lacZ )M15])
was used as host for all plasmid DNA manipulations and the
final expression and assay of gusA constructs. E . coli
RFS859 (F¹, thr -1, araC859 , leuB6 , Dlac74 , tsx -274, l¹,
gyrA111, recA11, relA1, thi -1) (Schlief, 1972) was used as
host for the final expression and assay of lacZ constructs.
E . coli BMH71-18 (mutS ::Tn10 , Dlac -proAB , supE , thi , [F8,
lacI q, lacZ , DM15-proA þB þ ]) (Novagen) was used in sitedirected mutagenesis using the unique site elimination
method (Deng and Nickoloff, 1992). Chromosomal DNA
from E . coli K-12 was used as the template for PCR amplification of the gusA gene. The plasmid pRSVCATam1.2.5 which
carries the U35A36 suppressor tRNA gene was kindly provided by Dr Uttam RajBhandary.
were purchased from Sigma. All other chemicals and reagents
were purchased from commercial sources and were of the
highest grade available. Oligonucleotides were synthesized
using a Beckman 1000 M oligo synthesizer. The lacZ specific
oligonucleotide 58-GGGGGATGTGCTGCAAGGCG-38 was
used in DNA sequencing, primer extension, and Northern
dot-blot hybridization and anneals to positions þ92 to þ73 of
the pUL-AUG lacZ CDS. The 16S rRNA-specific oligonucleotide
58-GGTTACCTTGTTACGACTTC-38 was used in Northern
dot-blot hybridization and anneals to positions þ1510 to
þ1491 of the E . coli 16S rRNA. The mutagenic oligonucleotide
58-CAGGAAACAGCCNTGGTTACGGATTC-38 (where N is
an equal mix of G, T and C) anneals to positions þ27 to
þ52 of the pSD-AUG lacZ and was used to mutagenize the
start codon. The mutagenic oligonucleotide 58-CGTATAATGTGTCCNTGGTTACGGATTC-38 (where N is an equal
mix of G, T, and C) anneals to positions ¹14 to þ14 of the
pUL-AUG lacZ and was used to mutagenize the start
codon. The oligonucleotides 58-AGTCCCCCATGGTACGTCCTGTCGACACCCC-38 and 58-GGAAGATCTCCCCCATTGCGAAGGC-38 anneal to positions þ75 to þ105 and
þ1978 to þ1954 of E . coli gusA (Genbank accession no.
S69414; Schlaman et al ., 1994), respectively, and were
used for cloning the gusA CDS from the E . coli K12 chromosome to the Nco I and Bgl II restriction sites within pIJ2920
(Janssen and Bibb, 1993) derivatives of pUL-AUG and pSDAUG, replacing the lacZ CDS to make pUL-AUGgus and
pSD-AUGgus. The start codons of pUL-AUGgus and pSDAUGgus were mutagenized by PCR using the oligonucleotide 58-CAGAATTCTGGCACGACAGGTTTCCCGACTGG-38
which anneals to ¹130 to ¹98 of the lac transcriptional start
site and the mutagenic oligonucleotide 58-GGGGTGTCGACAGGACGTACCANGGACAC-38 or 58-GGGGTGTCGACAGGACGTACCANGGCTGTTTCC-38 (where N is an equal
mix of G, T, and C) which anneals to þ61 to þ33 and þ23
to ¹10 of pSD-AUGgus and pUL-AUGgus gusA , respectively.
The gusA -specific oligonucleotide 58-CGCGATCCAGACTGAATGCCC-38 which anneals to þ76 to þ56 of pUL-AUGgus
was used for DNA sequencing and primer extension analysis.
Restriction endonucleases, T4 DNA ligase, T4 polynucleotide
kinase, and T4 DNA polymerase were obtained from New
England Biolabs and used according to the manufacturer’s
specifications. E . coli MR600 tRNA, AMV reverse transcriptase and RNase-free DNase I were purchased from Boehringer-Mannheim. Sequenase and Taq DNA polymerase were
purchased from United States Biochemical and used according to the manufacturer’s specifications. Plasmid DNA was
isolated by the alkaline lysis method (Sambrook et al .,
1989). Plasmid DNA used in dideoxy sequencing was further
purified using a Geneclean DNA purification kit (Bio 101).
Competent cell preparation and transformation was conducted by the CaCl2 method (Sambrook et al ., 1989). All
other DNA manipulations were carried out in the standard
manner (Sambrook et al ., 1989).
Reagents and recombinant DNA procedures
Radio-labelled nucleotides, [g-32P]-ATP (6000 Ci mmol¹1,
150 mCi ml¹1 ) and [a-32P]-dATP (3000 Ci mmol¹1, 10 mCi
ml¹1 ) were purchased from New England Nuclear. Isopropylthio-b-D-galactoside (IPTG), o -nitrophenyl-b-D-galactopyranoside (ONPG), and p-nitrophenyl-b-D-glucuronide (p-gluc)
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
Construction of leadered and unleadered lacZ with
NUG and UAG start codons
Seven site-directed mutants were constructed from plasmids
containing a lacZ reporter gene with (pSD-AUG), or without
(pUL-AUG), an untranslated leader (Janssen, unpublished
998 W. J. Van Etten and G. R. Janssen
results) (Fig. 1). These plasmids were constructed from the
pBR322 derivative pTL61T (Linn and St Pierre, 1990) and
contain strong transcriptional terminators flanking the leadered
and unleadered lacZ genes. The lacZ gene of pSD-AUG differs from wild type in the following manner with the following
justifications. The ¹10 region of the lac promoter was changed from TATGTT to TATAAT, creating a lacUV5 -10 region,
for its ability to maintain the transcriptional start site independent of downstream sequences (Lorimer et al ., 1990) and to
minimize catabolite regulation (Silverstone et al ., 1970). The
untranslated leader and CDS were altered from wild type
within the ¹2 to þ12 positions, relative to the translational
start site, from ctATG·ACC·ATG·ATT to ccATG·GTT, deleting the second and third codons (underlined) which eliminate
the secondary translational start site (Munson et al ., 1984)
and create an Nco I (58-CCATGG-38) restriction site. The
unleadered lacZ reporter plasmid, pUL-AUG, was constructed
from pSD-AUG by deletion of the 38 nucleotides preceding
the Nco I restriction site such that the transcriptional and
translational start sites are coincident.
The Eco RI– Pvu II fragments of pSD-AUG and pUL-AUG
were subcloned into high copy vectors for use as templates
for oligonucleotide site-directed mutagenesis by either the
unique restriction site elimination (Deng and Nickoloff, 1992)
or the double PCR (Kim et al ., 1996) methods. Plasmid templates were mutagenized with oligonucleotides (see Reagents
and recombinant DNA procedures ) that change the start
codon within the unique Nco I restriction site. Mutant plasmids
were selected by restriction with Nco I and transformed into E .
coli DH5a. To ensure isogenicity of the final mutant clones,
the Eco RI– Pvu II fragments from transformants that had lost
the unique Nco I site were sequenced. The resulting mutant
Eco RI– Pvu II fragments were subcloned back into the fulllength lacZ genes within pSD-AUG and pUL-AUG, then transformed into the lacZ deletion strain E . coli RFS859. In addition,
the lacZ genes and flanking transcriptional terminators from
pSD-AUG, pSD-UAG, pUL-AUG, pUL-UAG and pUL-UUAG
were independently cloned into pACYC171 and subsequently
transformed into RFS859 harbouring a compatible pIJ2920derived plasmid with (pAmSUPþ) or without (pAmSUP¹)
an amber-suppressor initiator tRNA. The plasmid pAmSUPþ
was cloned by inserting the Bam HI– Pst I restriction fragment
from pRSVCATam1.2.5 (Varshney and RajBhandary, 1990),
carrying the U35A36 tRNA gene, into the same sites within
pIJ2920 (Janssen and Bibb, 1993). The negative control plasmid pAmSUP¹ was constructed through the deletion of the
multiple cloning site within a Pvu II restriction fragment and
the ligation of the resulting vector fragment.
The start codons of pUL-AUGgus and pSD-AUGgus were
mutagenized by PCR using a 58 oligonucleotide (see Reagents
and recombinant DNA procedures ) containing an Eco RI
restriction site, which anneals upstream of the lacUV5 promoter, and 38 mutagenic oligonucleotides that includes the
Sal I site and introduces the single base changes at the þ1
position of the start codon. The resulting mutant Eco RI–
Sal I restriction fragments were cloned back into pULAUGgus and pSD-AUGgus. Resulting clones were screened
for the loss of the unique Nco I restriction site and sequence
verified through the subcloned Eco RI– Sal I fragment. The
plasmid pUL-10mtgus was constructed by replacing the
Eco RI– Nco I promoter fragment with the same from Plac *
(Wu and Janssen, 1996).
Construction of leadered and unleadered cI– lacZ
fusions with NUG and UAG start codons
PCR amplification was used to clone bacteriophage lambda
c I codons 1–16 between the Eco RV and Sal I sites of a lacZ
translational fusion plasmid where the Eco RV site maps to
the lacUV5 transcriptional start site and the Sal I site is located
at lacZ codon five. PCR amplification with Pfu DNA polymerase
(Stratagene), pIU1041 (Wu and Janssen, 1997) as template,
a downstream primer (58-ACGCTCATCGATAATTTCACCGCC-38), and each of the following upstream primers was
used to prepare blunt-ended DNA fragments of c I– lacZ with
the indicated 58 terminal start codons: AUG: 58-ATGAGCACAAAAAAGAAACCATTAAC-38; UAG: 58-TAGAGCACAAAAAAGAAACCATTAAC-38; UAUAG: 58-TATAGAGCACAAAAAAGAAACCATTAAC-38. The reaction products were
cloned separately into a plasmid containing a lacUV5 promoter such that transcription was expected to initiate at the first
position of the cloned fragment or a plasmid where the cloning
site mapped to the translational start site of a modified leadered lacZ . The amplified region was subjected to DNA
sequencing to ensure presence of the desired mutation and
the absence of unwanted secondary site changes. The resulting mutant Eco RI– Sac I fragments, containing the lacUV5
promoter region, c I codons 1–16, and a segment of lacZ
CDS were then subcloned back into the full-length lacZ
genes within pSD-AUG and pUL-AUG. The c I– lacZ fusions
and flanking transcriptional terminators from pSD-AUGcI,
pSD-UAGcI, pUL-AUGcI, pUL-UAG, and pUL-UAUAGcI
were independently cloned into pACYC177 and subsequently
transformed into RFS859 harbouring a compatible pIJ2920derived plasmid with (pAmSUPþ) or without (pAmSUP¹)
an amber-suppressor initiator tRNA.
Construction of leadered and unleadered gusA with
NUG start codons
b-Galactosidase activity measurements
The b-glucuronidase (gusA ) CDS was amplified from E . coli
K-12 chromosomal DNA by PCR using oligonucleotides
(see Reagents and recombinant DNA procedures ) that introduce an Nco I restriction site at the initiation codon, a Sal I
restriction site at codon five of the gusA CDS, and a Bgl II
restriction site downstream of the CDS. The resulting Nco I–
Bgl II restriction fragment was cloned into pIJ2920 (Janssen
and Bibb, 1993) derivatives of pUL-AUG and pSD-AUG, replacing the lacZ CDS, to make pUL-AUGgus and pSD-AUGgus.
E . coli RFS859 containing the individual leadered and unleadered lacZ constructs were grown to an OD600 of 0.3–0.6 in
triplicate 50 ml cultures of 2XYT (16 g l¹1 Bacto-tryptone,
10 g l¹1 Bacto-yeast extract, 10 g l¹1 NaCl, pH 7.4) supplemented with IPTG (0.2 mM) and antibiotics where appropriate
(200 mg ml¹1 ampicillin or 50 mg ml¹1 kanamycin) at 378C then
quick-chilled on ice. Triplicate b-galactosidase assays were
performed on each of the triplicate cultures according to the
method of Miller (1992).
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 999
b-Glucuronidase activity measurements
Determination of translational efficiency
As in the b-galactosidase assays, cells were grown to an
OD600 of 0.3–0.6 in triplicate 50 ml cultures of 2XYT supplemented with IPTG (0.2 mM) and antibiotic when appropriate
(200 mg ml¹1 ampicillin) at 378C then quick-chilled on ice. Triplicate b-glucuronidase assays were performed on each of
the triplicate cultures by a modified b-galactosidase assay
derived from the method of Miller (1992) with the following
modifications: the chromogenic substrate, p-gluc, was used
at 2 mg ml¹1 in place of ONPG, and the extent of p-gluc hydrolysis was monitored by absorbance using a wavelength of
415 nm rather than 420 nm. The formula used to quantify
b-glucuronidase activity was: (OD415 × 1000)/(OD600 ×
volume, ml × time, min).
Translational efficiency was computed as the quotient of the
b-galactosidase activity (Miller units) and the normalized
lacZ -specific hybridization signal [mean of the lacZ -specific
hybridization signal (cpm) divided by the mean of the 16S
rRNA-specific hybridization signal (cpm)].
RNA preparation
Total RNA was isolated from two of the three cultures used in
the b-galactosidase assays as described previously (Wu and
Janssen, 1996).
Radio-labelling of oligonucleotides
The lacZ and 16S rRNA-specific oligonucleotides (30 pmol)
were end-labelled with 225 mCi of [g-32P]-ATP and 10 U of
T4 polynucleotide kinase in a 10 ml buffered reaction [20 mM
Tris-HCl (pH 8.0), 10 mM magnesium acetate, 10 mM DTT]
at 378C for 30 min. End-labelled oligonucleotides were purified on Pharmacia Sephadex G50 Nick columns according
to the manufacturer’s specifications.
Primer extension analysis
Primer extension reactions containing 40 mg of RNA and
2 pmol of end-labelled lacZ specific oligonucleotide were performed as described previously (Brown et al ., 1988). Equal
volumes of each reaction were electrophoresed against
appropriate dideoxy sequencing reactions, and visualized by
autoradiography.
Northern dot-blot analysis
The Northern dot-blot was prepared on a Schleicher & Schuell
Nytran filter membrane (0.2 mm pore size) according to the
method of Schleicher & Schuell (Schleicher and Schuell,
1987) with the following modifications and additions. Aliquots
of total cellular RNA (0, 1, 2, 4, 6 mg) were supplemented with
E . coli tRNA to a total mass of 6 mg before loading onto the
membrane. The lacZ -specific end-labelled oligonucleotide
probe was used at 106 cpm ml¹1 hybridization solution, and
was hybridized and washed at 688C. The 16S rRNA specific
end-labelled oligonucleotide probe was diluted (3:160) with
unlabelled oligonucleotide and used at an estimated 10-fold
molar excess of annealing sites (assuming rRNA is 80% of
total RNA and each of the rRNAs are present in equimolar
amounts). The hybridization and washing temperature used
for the 16S rRNA-specific probe was 538C. The lacZ and
16S rRNA hybridizations were quantified using an Ambis
2000 beta-scanner.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
Acknowledgements
We thank JR Liston and Julie Martin-Farmer for reviewing the
manuscript. We also thank Dr Uttam RajBhandary for the
plasmid pRSVCATam1.2.5 carrying the U35A36 suppressor
tRNA gene, Tim Fitzwater for E . coli BMH71-18, and Luis
Actis for the plasmid pACYC177. We thank Miami University
MBI465 Molecular Genetics Laboratory class for E . coli K-12
DNA. This research was supported by the National Institutes
of Health Grant GM45923.
References
Balakin, A.G., Skripkin, E.A., Shatsky, I.N., and Bogdanov,
A.A. (1992) Unusual ribosome binding properties of
mRNA encoding bacteriophage l repressor. Nucleic
Acids Res 20: 563–571.
Bibb, M.J., White, J., Ward, J.M., and Janssen, G.R. (1994)
The mRNA for the 23S RNA methylase encoded by the
ermE gene of Saccharopolyspora erythraea is translated
in the absence of a conventional ribosome-binding site.
Mol Microbiol 14: 533–545.
Boni, I.V., Isaeva, D.M., Musychenko, M.L., and Tzareva,
N.V. (1990) Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1. Nucleic Acids Res 19:
155–162.
Brown, J.W., Thomm, M., Beckler, G.S., Frey, G., Stetter,
K.O., and Reeve, J.N. (1988) An archaebacterial RNA
polymerase binding site and transcription initiation of the
hisA gene in Methanococcus vanniellii. Nucleic Acids
Res 16: 135–150.
Childs, J., Villanueba, K., Barrick, D., Schneider, T.D.,
Stormo, G., Gold, L. et al . (1985) Ribosome binding sites
sequences and function. In Sequence Specificity in Transcription and Translation . Calendar, R., and Gold, L
(eds). New York: Alan R. Liss, pp. 341–350.
Deng, W.P., and Nickoloff, J.A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique
site. Anal Biochem 200: 81–88.
Gold, L. (1988) Postranscriptional regulatory mechanisms in
Escherichia coli . Annu Rev Biochem 57: 199–233.
Gualerzi, C.O., and Pon, C.L. (1990) Initiation of mRNA
translation in prokaryotes. Biochemistry 29: 5881–5889.
Hartz, D., Binkley, J., Hollingsworth, T., and Gold, L. (1990)
Domains of initiator tRNA and initiation codon crucial for
initiator tRNA selection by Escherichia coli IF3. Genes
Dev: 1790–1800.
Hoshiko, S., Nojiri, C., Matsunaga, K., Katsumata, K., Satoh,
E., and Nagaoka, K. (1988) Nucleotide sequence of the
ribostamycin phosphotransferase gene and of its control
region in Streptomyces ribosidificus . Gene 68: 285–296.
Hui, A., and de Boer, H.A. (1987) Specialized ribosome system: preferential translation of a single mRNA species by a
1000 W. J. Van Etten and G. R. Janssen
subpopulation of mutated ribosomes in Escherichia coli .
Proc Natl Acad Sci USA 84: 4762–4766.
Jacob, W.F., Santer, M., and Dahlberg, A.E. (1987) A single
base change in the Shine–Dalgarno region of 16S RNA of
Escherichia coli affects translation of many proteins. Proc
Natl Acad Sci USA 84: 4757–4761.
Janssen, G.R. (1993) Eubacterial, archaebacterial, and
eucaryotic genes that encode leaderless mRNA. In Industrial Microorganisms: Basic and Applied Molecular Genetics . Baltz, R.H., Hegeman, G.D., and Skatrud, P.L. (eds.)
Washington, DC: American Society for Microbiology, pp.
59–67.
Janssen, G.R., and Bibb, M.J. (1993) Derivatives of pUC18
that have Bgl II sites flanking a modified multiple cloning
site and that retain the ability to identify recombinant
clones by visual screening of Escherichia coli colonies.
Gene 124: 133–134.
Janssen, G.R., Ward, J.M., and Bibb, M.J. (1989) Unusual
transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph ) from Streptomyces
fradiae . Genes Dev 3: 415–429.
Jones, III, R.L., Jaskula, J.C., and Janssen, G.R. (1992) In
vivo translational start site selection on leaderless mRNA
transcribed from Streptomyces fradiae aph gene. J Bacteriol 174: 4753–4760.
Kim, J., Puder, M., and Soberman, R.J. (1996) Joining of
DNA fragments by repeated cycles of denaturation, annealing and extension. Biotechniques 20: 954–955.
Linn, T., and St. Pierre, R. (1990) Improved vector system for
constructing transcriptional fusions that ensures independent translation of lacZ . J Bacteriol 172: 1077–1084.
Lorimer, D.D., Cao, J., and Revzin, A. (1990) Specific
sequences downstream from ¹6 are not essential for
proper and efficient in vitro utilization of the Escherichia
coli lactose promoter. J Mol Biol 216: 275–287.
Miller, J.H. (1992) A Short Course in Bacterial Genetics: a
Laboratory Manual and Handbook for Escherichia coli
and Related Bacteria . Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Munson, L.M., Stormo, G.D., Niece, R.L., and Reznikoff,
W.S. (1984) lacZ translation initiation mutations. J Mol
Biol 177: 663–683.
Noll, H., Noll, M., Hapke, B., and van Dieijen, G. (1973) In
Regulation of Transcription and Translation, Mosbach
Colloqium no. 24 . Heidelberg: Springer, pp. 257–311.
Olins, P.O., and Rangwala, S.H. (1989) A novel sequence
element derived from bacteriophage T7 mRNA acts as
an enhancer of the lacZ gene in Escherichia coli . J Biol
Chem 264: 16973–16976.
Ptashne, M., Backman, K., Humayum, M.Z., Jeffrey, A.,
Maurer, R., Meyer, B., and Sauer, R.T. (1976) Autoregulation and function of a repressor in bacteriophage lambda.
Science 194: 156–161.
RajBhandary, U.L., and Chow, C.M. (1995) Initiator tRNAs
and initiation of protein synthesis. In tRNA: Structure,
Biosynthesis, and Function . Soll, D., and RajBhandary,
U.L. (eds.) Washington, DC: American Society for Microbiology.
Ray, P.N., and Pearson, M.L. (1974) Evidence for post-transcriptional control of the morphogenetic genes of bacteriophage lambda. J Mol Biol 85: 163–175.
Ray, P.N., and Pearson, M.L. (1975) Functional inactivation
of bacteriophage l morphogenetic gene mRNA. Nature
253: 647–650.
Ringquist, S., Shinedling, S., Barrick, D., Green, L., Binkley,
J., Stormo, G.D., and Gold, L. (1992) Translation initiation
in Escherichia coli : sequences within the ribosome-binding
site. Mol Microbiol 6: 1219–1229.
Risuleo, G., Gualerzi, C., and Pon, C. (1976) Specificity and
properties of the destablization, induced by initiation factor
IF-3, of ternary complexes of the 30-S ribosomal subunit,
aminoacyl-tRNA and polynucleotides. Eur J Biochem 67:
603–613.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual – 2nd edn. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
Scherer, G.F.E., Walkinshaw, M.D., Arnott, S., and Mooré,
D.J. (1980) The ribosome binding sites recognized by E.
coli ribosomes have regions with signal character in both
the leader and protein coding segments. Nucleic Acids
Res 8: 3895–3907.
Schlaman, H.R., Risseeuw, E., Franke-van Dijk, M.E., and
Hooykaas, P.J. (1994) Nucleotide sequence corrections
of the uidA open reading frame encoding beta-glucuronidase. Gene 138: 259–260.
Schleicher and Schuell. (1987) Transfer and Immobilization
of Nucleic Acids to S&S Solid Supports . USA: Schleicher
& Schuell.
Schlief, R. (1972) Fine-structure deletion map of the Escherichia coli L-arabinose operon. Proc Natl Acad Sci USA 69:
3479–3484.
Schneider, T.D., Stormo, G.D., and Gold, L. (1986) Information content of binding sites on nucleotide sequences. J
Mol Biol 188: 415–431.
Shine, J., and Dalgarno, L. (1974) The 38 terminal of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad
Sci USA 71: 1342–1346.
Silverstone, A.E., Arditti, R.R., and Magasanik, B. (1970)
Catabolite-insensitive revertants of lac promoter mutants.
Proc Natl Acad Sci USA 66: 773–778.
Sprengart, M.L., Fatscher, H.P., and Fuchs, E. (1990) The
initiation of translation in E. coli : apparent base pairing
between the 16S RNA and downstream sequences of the
mRNA. Nucleic Acids Res 18: 1719–1723.
Sprengart, M.L., Fuchs, E., and Porter, A.G. (1996) The
downstream box: an efficient and independent translation
signal in Escherichia coli . EMBO J 15: 665–674.
Steitz, J.A. (1969) Polypeptide chain initiation: nucletide
sequences of the three ribosomal binding sites in bacteriophage R17 RNA. Nature 224: 957–964.
Steitz, J.A., and Jakes, K. (1975) How ribosomes select
initiator regions in mRNA: base pair formation between
the 38 terminus of 16S RNA and the mRNA during initiation
of protein synthesis in Escherichia coli . Proc Natl Acad Sci
USA 72: 4734–4738.
Steitz, J.A., and Steege, D.A. (1977) Characterization of two
mRNA: rRNA complexes implicated in the initiation of protein biosynthesis. J Mol Biol 114: 545–558.
Stoekenius, W., Lozier, R.H., and Bogomolni, R.A. (1979)
Bacteriorhodopsin and the purple membrane of halobacteria. Biochim Biophys Acta 505: 215–278.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
AUG start codon and the translation of unleadered mRNA 1001
Stormo, G.D., Schneider, T.D., and Gold, L.M. (1982) Characterization of translational initiation sites in E. coli . Nucleic
Acids Res 10: 2971–2996.
Sussman, J.K., Simons, E.L., and Simons, R.W. (1996)
Escherichia coli translation initiation factor 3 discriminates
the initiation codon in vivo . Mol Microbiol 21: 347–360.
Tzareva, N.V., Makhno, V.I., and Boni, I.V. (1994) Ribosomemessenger recognition in the absence of the Shine–
Dalgarno interactions. FEBS Lett 337: 189–194.
van Wezel, G.P., White, J., Young, P., Postma, P.W., and
Bibb, M.J. (1997) Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2)
is controlled by malR , a member of the lacI-galR family of
regulatory genes. Mol Microbiol 23: 537–549.
Varshney, U., and RajBhandary, U.L. (1990) Initiation of protein synthesis from a termination codon. Proc Natl Acad
Sci USA 87: 1586–1590.
Vollenoweth, R.l., and Rabinowitz, J.C. (1992) The influence
of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo .
Mol Microbiol 6: 1105–1114.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 987–1001
Wu, C.-J., and Janssen, G.R. (1996) Translation of vph
mRNA in Streptomyces lividans and Escherichia coli after
removal of the 58 untranslated leader. Mol Microbiol 22:
339–355.
Wu, C.-J., and Janssen, G.R. (1997) Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli . J Bacteriol 179: 6824–
6830.
Wu, X.-Q., and RajBhandary, U.L. (1997) Effect of the amino
acid attached to Escherichia coli initiator tRNA on its
affinity for the initiation factor IF2 and on IF2 dependence
of its binding to the ribosome. J Biol Chem 272: 1891–
1895.
Wu, X.-Q., Iyengar, P., and RajBhandary, U.L. (1996) Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant
initiator tRNAs and specialized ribosomes. EMBO J 15:
4734–4739.
Zhang, J., and Deutscher, M.P. (1991) A uridine-rich sequence required for translation of prokaryotic mRNA. Proc
Natl Acad Sci USA 89: 2605–2609.