TRANSLATIONAL COUPLING DURING EXPRESSION OF THE
TRYPTOPHAN OPERON OF ESCHERZCHZA COLZ
DANIEL S . OPPENHEIM AND CHARLES YANOFSKY
Department of Biological Sc:ences, Stanford University, Stanford, CA 94301
Manuscript received February 26, 1980
ABSTRACT
E. coli trpE polar mutations are 10 times more polar on trpD gene expression than on downstream (trpC, B, or A ) gene expression. This effect was
shown to be the result of "translational coupling," in which efficient translation of trpD mRNA reqiures efficient translation of the end of trpE mRNA.
The trpE-trpD intercistronic punctuation region consists of overlapping stop
and start codons, and the trpE and trpD gene products form a functional
complex in the cell. In light of these observations and characteristics, several
models for the mechanism of translational coupling are considered.
HE tryptophan ( t r p ) operon of Escherichia coli consists of a cluster of five
Tgenes whose products catalyze the final steps in the biosynthesis of the amino
acid tryptophan (Figure 1) (YANOFSKY
1971). Normally, the five gene products
are synthesized in equimolar amounts from polycistronic messenger RNA molecules ( IMAMOTO
and YANOFSKY
1967a). Mutations in one of the first four genes of
the operon that cause polypeptide chain termination may reduce the expression of
downstream genes (YANOFSKY
and ITO
1966; YANOFSKY
et al. 1971). This mutational polarity is thought to be due to transcription termination, mediated by rho
FIGURE
1.-Genes of the tryptophan operon of E. coli, their polypeptide products and reactions catalyzed by polypeptides or polypeptide complexes. p-o denotes the trp promoter-operator
region; a, the trp attenuator; and p2, the low-level internal promoter. Numbers indicate positions
of mutations used in this study. Direction of transcription is from left to right. CoI and CoII
signify components I and I1 of anthranilate synthetase complex, respectively. PR anthranilate,
N-5'-phosphoribosyl-anthranilate;CdRP, I-(0-carboxyphenylamin0)-I-deoxyribulose-5-phosphate
;
InGP, indole-3-glycerol phosphate; PRPP, 5-phosphoribosyl-I pyrophosphate.
Genetics 95 : 785-795 August, 1980.
786
D. S .
OPPENHEIM A N D C. YANOFSKY
factor, in the region between the nonsense codon and the beginning of the next
gene (ADHYA
and GOTTESMAN
1978). Thus, in a strain bearing a polar mutation,
the amount of mRNA homologous to genes downstream from the mutated gene
is reduced (IMAMOTO
and YANOFSKY
1976a, b). All segments of mRNA from
genes downstream from the mutated gene are reduced to the same extent and
have normal stability (MORSEand YANOFSKY
1969; HIRAGA
and YANOFSKY
197213).
Mutational polarity can be partially relieved by the introduction of either of
two types of suppressor mutations. Mutations affecting rho factor suppress polarity by reducing transcription termination, thereby restoring downstream mRNA
synthesis, while leaving the mRNA corresponding to the distal segment of the
mutated gene untranslated (KORNand YANOFSKY
1976b). Polarity is also relieved
by nonsense suppressors that allow translation at and beyond the mutant site
(YANOFSKY
and ITO1966) . Presumably, whenever the mutant site is translated,
rho-mediated transcription termination is prevented, and downstream mRNA
levels are elevated concomitant with translation of the 3’ end of the mutated
gene.
et al. (1971) observed that nonI n studying mutational polarity, YANOFSKY
sense mutations in trpE are considerably more polar on the expression of trpD
than they are on trpB or A. They presented evidence suggesting that this disprportionality was not due to a decrease in the activity or stability of the trpD
protein in the absence of its normally associated polypeptide subunit, the trpE
protein.
I n the present study, we investigate the cause of the disproportionately low
expression of trpD in TrpE polar mutants by examining gene expression, measured as trp enzyme activities, in trpE polar mutants in the presence of either of
the two types of polarity-relieving suppressors described above. We find that rhodefective polarity suppressors, which are believed to elevate downstream mRNA
levels but leave the distal portion of trpE mRNA untranslated, maintain the
disproportionately low expression of trpD. In contrast, trpD expression is nearly
equal to that of downstream genes when polarity is relieved by nonsense suppressors that allow translation to the end of trpE mRNA. This suggests that the
normally efficient translation of trpD mRNA depends on the translation of the
eed of trpE mRNA. Thus, there is an apparent “translational coupling” between
trpE and trpD expression. We also examine other genes in the trp operon for
this translational coupling.
M A T E R I A L S A N D METHODS
Bacterial struins: All irp amber mutations were introduced into the same W3110 trpR lac2
U118 ValR AziR Rho+ background or is rho102 or rho103 (KORN and YANOFSKY
1976a, b)
derivative by PI clr transduction. Nonsense suppressors were introduced from W3110 Arton.
BtrpAEl] sup+ strains except in the case of trpE9828 supF, which is lysogenic for $80 carrying
supF. pPSS1, a plasmid carrying the trp regulatory region and trpE+, but lacking most of the
trpD gene (P.STORK and C. YANOFSKY,
unpublished), was introduced into the trpE9780 strain
and
by transformation. trpAE5 is an internal deletion removing almost all of trpE (HIRAGA
787
TRANSLATIONAL C O U P L I N G
YANOFSKY1972a). The trpALE1413 deletion removes the trp attenuator and the initial 60%
Qf trpE (MIOZZARI
and YANOFSKY
1978).
Media and enzyme assays: 200 ml cultures were grown for enzyme assays in minimal medium
(VOGEL
and BONNER
1956) supplemented with 0.2% glucose, 0.05% acid hydrolyzed casein and
50 pg/ml L-tryptophan. At a density of -6 x 10s cells/ml, cells were collected by centrifugation, washed in N ml cold 0.85% NaC1, resedimented and resuspended in 2.0 ml cold 0.1 M
tris HC1 buffer, pH 7.8. Extracts were prepared by sonic disruption, followed by removal of
cell debris by centrifugation at 17,000 x g for 15 min. trpE (chorismate NH, + anthranilate), trpD (anthranilate f- PRPP + PR anthrani1ate)l and trpC (CdRP + InGP) enzyme
activities were assayed as described by CREIGHTONand YANOFSKY
(1970). trpB and tip4 polypeptides were assayed in the indole serine + tryptophan reaction as described by SMITH
and YANOFSKY(1962). Protein concentration was determined by the method of LOWRY
et al.
(1951).
+
+
RESULTS
trpE polar nutations: In trpE polar mutants, is trpD expression reduced relative to downstream gene expression because the end of trpE mRNA is untranslated? To answer this question, we compared trp enzyme specific activities in
extracts of strains carrying trpE amber polar mutations with and without either
the polarity suppressors, rhol02 or rhol03, or the amber suppressors, supD or
supF. Table 1 summarizes the results for both moderately strong (trpE9780) and
very strong (trpE9829) polar mutations. These mutations are considerably
more polar on trpD expression than on distal (frpC,B or A ) gene expression, as
can be seen from the distal-to-D ratios (e.g., 7- to 8-fold more polar on D than
A expression). The introduction of either polarity suppressor, rho102 or rh0.203,
TABLE 1
Eflecr of trpE polar mutations
Strain
D
trpE9780 rho+ sup+
rho102
rho103
supD
supF
1.5
6.2
5.0
12
14
trpE9829 rho+ sup+
rho102
rho103
supF
0.19
5.4
6.2
25
Enzyme specific activity (% of Trp+control cultures)
C
B
A
CID
BiD
8
28
20
22
37
15
56
53
38
38
12
50
63
29
33
1.4
38
35
36
5.3
4.5
4.0
1.8
2.6
10
9.0
11
3.1
2.7
AiD
8.0
8.1
13
2.4
2.4
7.4
7.0
5.6
1.4
All strains contain the same trpR mutation. Control cultures were W3110 trpR lac2 U118
ValR AziR Trp+ rho+ Sup+. Growth conditions and reactions assayed are described in MATERIALS
AND METHODS. Enzyme abbreviations: D = phosphoribosyl anthranilate transferase, C =
indolyl-3-glycerol phosphate synthetase; B = tryptophan synthetase p2; A = tryptophan
synthetase a. To compensate for the lonr level expression from the internal promoter p., a value
of 4% was subtracted from all C, B, and A values (JACKSONand YANOFSKY1972). The effect
of the rho mutations at the trp attenuator (KORNand YANOFSKY
1976a) is, in our hands, variable, but always less than 2-fold. In any case, consideration of specific activity ratios obviates
this concern.
1
See Figure 1 for abbreviations
788
D. S. OPPENHEIM A N D C. Y A N O F S K Y
substantially relieves the polar effect of the trp mutations (3- to 4-fold relief in
trp 9780 and 25- to 30-fold relief in trpE9829). However, this relief is about the
same for trpD and distal gene expression. The disproportionately low trpD
expression is maintained and the distal-to-D ratios are about the same in the
presence or absence of the rho polarity suppressors.
The nonsense suppressors, supD and supF, also relieved the polar effect of the
trpE mutations. Their effect on D-distal expression is in the same range as the
effect of the rho-defective polarity suppressors. However, the nonsense suppressors elevate trpD enzyme levels to a greater extent (8- to 9-fold for trpE9780
and 130-fold for trpE9829) than they elevate downstream enzyme levels, thus
substantially reducing the distal-to-D ratios. That is, the two types of suppressors
have different effects on trpD expression; whereas, they restore trpC, B and A
protein production nearly equally. Polarity suppressors, which leave the end of
trpE mRNA untranslated, maintain the disproportionately low trpD expression.
Nonsense suppressors, which allow trpE mRNA translation to be completed,
elevate trpD expression more than trpC, B or A expression, restoring a closer to
wild-type ratio of distal to D expression. This result suggests that translation of
the end of trpE mRNA is required for normal, efficient expression of trpD.
To reconfirm that the absence of trpE protein itself is not the cause of the
nonequivalent polarity described above and that the effect seen with the nonsense suppressors is not simply due to the restoration of trpE protein, we introduce the trpE+ trpD- plasmid pPS21 into the trpE9780 strain to provide a source
of trpE protein. The results in Table 2 show that despite the presence of a substantial level of trpE protein, trpD expression is still reduced relative to trpA.
Table 2 also shows enzyme activities in two trpE deletion strains. trpAE5 covers
all known trpE point mutations, yet is not polar on downstream genes (HIRAGA
and YANOFSKY
1972a). Therefore, this deletion is presumed to be totally within
trpE, removing almost all of the trpE sequence. We assume that translation of the
remaining portions at the very beginning and end of the E gene occurs in the
normal phase and at the normal rate. The fact that trpD expression in this deletion strain is the same as in the trp+ control supports the argument that it is not
the presence of trpE protein that maintains trpD expression at its wild-type level.
TABLE 2
Effect of trpE muiations
~~
Enzyme specific activity
Strain
frpE9780
frpE9780/pPSZl
trpAE5
trpALEl413
E
D
A
AID
0
1.5
2.7
110
12
13
1(15
680
4.8
0.95
54
See notes for Table 1. E =anthranilate
described in the text.
(xof Trp+control cultures)
1200
8
0.57
synthetase Component I. Strains employed are
789
TRANSLATIONAL COUPLING
TABLE 3
Effect of trpD polar mutations
Strain
C
Specific activity ( % of Trp+ control cultures)
B
B/C
supD
1.6
28
31
15
supF
26
trpD.249 rho+ sup
rho102
rho103
4.1
72
76
26
30
2.6
2.6
2.5
1.7
1.2
See notes for Table 1.
Similar considerations apply to deletion strain trpnLE1413, which has a deletion removing the trp attenuator and the first 600/;,of trpE (MIOZZARI
and YANOFSKY 1978). This deletion is known to be nonpolar and to fuse the remaining
portion of trpE in phase to the trp leader polypeptide coding region. This deletion
results in the production of a fusion polypeptide containing an amino-terminal
sequence specified by the leader region and the remainder by the distal segment
of trpE. That both D and A protein levels are substantially increased (due to
removal of the attenuator) supports the view that the disporportionately low
trpD expression seen in trpE polar mutants is not due to the absence of the trpE
protein. It is interesting that upon removal of the trp attenuator, trpD expression
is elevated almost two-fold more than trpA expression. If trpD mRNA translation
is coupled to trpE mFWA translation, this two-fold difference could reflect more
efficient translation from the trp leader peptide translation start site than from
the trpE translation start site.
trpD and trpC polar mutations: For the sake of comparison and correlation
with other features of the trp operon, it was of interest to investigate the effect
of polar mutations in the other trp genes on coordinate translation of downstream
genes. Table 3 shows the results of a similar analysis with a strong polar trpD
nonsense mutation. The data show that trpD249 is about two- to three-fold more
polar on trpC than on trpB expression (or on trpA; data not shown). Polarity
suppressors maintain this difference, while nonsense suppressors largely eliminate this difference.
TABLE 4
Effect of trpC polar mutations
Strain
trpC10295 rho+ sup+
rho102
rho103
supD
supF
See nates for Table 1.
Specific activity ( % of Trp+control cultures)
B
A
B/A
19
63
89
43
26
15
53
82
29
30
1.3
1.2
1.1
1.5
1.2
790
D. S. O P P E N H E I M A N D C. Y A N O F S K Y
Table 4 indicates that a trpC nonsense mutation is equally polar on trpB and
t r p A expression. This equality of expression persists when polarity is relieved
by either of the two types of suppressors.
DISCUSSION
Enzyme-specific activities were determined in extracts of strains bearing t r p
polar mutations and either of two types of polarity suppressing mutations: rhodefective “polarity suppressors,” which relieve polarity without restoring translation of the mRNA segment corresponding to the terminal portion of the mutated
trp gene, or tRNA-altered “nonsense suppressors,” which relieve polarity b y
allowing such translation. We have shown that the disproportionately low expression of the trp-D gece relative to D-distal genes seen in two trpE polar mutants
persists in the presence of the polarity suppressors, rho102 and rho103, but is
largely reduced by the nonsense suppressors, supD and supF. Thus, it appears
that allowing translation of trpE mRNA increases trpD enzyme activity to nearnormal levels relative to downstream gene expression.
I n a trpE polar mutant, trpD mRNA and mRNA homologous to distal t r p
genes are reduced to the same extent (MORSE
and YANOFSKY
1969; HIRAGA
and
YANOFSKY
1972b). Therefore, the greater reduction of trpD activity in a trpE
polar mutant must be due either to decreased translation of trpD mRNA or to
decreased stability or activity of the trpD protein in the absence of trpE expression. Conversely, the disproportionate increase in trpD activity in the presence
of a nonsense suppressor must be due either to the presence of the trpE polypeptide o r to increased translation per se of trpE mRNA.
Even in the presence of substantial amounts of trpE protein produced from a
trpE-bearing plasmid, trpD expression is still low. Furthermore, deletions that
remove much or nearly all of the trpE gene, but presumably allow normal translation of the remaining trpE mRNA sequences, do not decrease relative trpD
expression. These results indicate that it is not the absence of the trpE polypeptide, but the absence of trpE translation per se that causes the disproportionately
low trpD expression.
The results presented here strongly suggest that the efficient translation of a t
least one gene ( t r p D ) in the polycistronic trp operon is dependent on the translation of the immediately preceding gene ( t r p E ) . We call this dependency
c<
translational coupling.” Translational coupling is clearly not operating between
at least one other pair of genes: trpC and trpB.
Two models for the mechanism of direct translational coupling come to mind.
Both are based on the premise that the trpD translation start site is, in the absence
of trpE translation, a relatively poor binding or initiation site for ribosomes:
(a) In the absence of trpE mRNA translation, the messenger RNA exists in a
conformation that disallows efficient ribosome binding and translation initiation
at the trpD start site. The progress of ribosomes along trpE mRNA alters the
secondary or tertiary structure of the messenger so that efficient initiation at
the trpD ribosome binding site is now possible. (b) The same ribosome or a com-
TRANSLATIONAL COUPLING
791
ponent of the same ribosome (e.g., the 30s subunit) that translates trpE mRNA
also translates trpD mRNA. That is, a ribosome binding and initiating de novo
at the trpD translation start site does so poorly, but a ribosome already on the
message will, upon termination of trpE mRNA translation, efficiently initiate
trpD mRNA translation. The corollary of both models is that these considerations
do not apply to the trpB translation start site.
A third possibility is that the coupling is indirect. For example, in the absence
of complete trpE mRNA translation, the trpD translation initiation site could
somehow be inactivated, e.g., by nucleolytic attack (see LIM and KENNELL
1979). It is interesting that even though trpD mRNA appears to be translated at
only 15% of its normal efficiency in a trpE polar mutant, such a mutant exhibits
none of the symptoms of a trpD polar mutant; trpD mRNA is as stable and as
and YANscarce as downstream mRNA (MORSE
and YANOFSKY
1969; HIRAGA
OFSKY 1972b). That is, a dramatic decrease in the number of ribosomes translating trpD mRNA appears to leave this RNA no more vulnerable to degradation
or to rho-mediated transcription termination.
Gene expression in the RNA bacteriophages is controlled in part by ribosomeeffected alterations in RNA secondary structure (STEITZ1975). The replicase
ribosome binding site is masked by base pairing with sequences in the coat protein gene. Only after ribosomes translating the coat protein gene have disrupted
this secondary structure can the translation of the replicase gene be initiated.
In this connection, we have examined the region from 100 nucleotides before
to 100 nucleotides after the trpE-trpD boundary for dyad symmetry (KORN,
QUEENand WEGMAN
1977). One region of secondary structure revealed by this
computer analysis is the stem and loop noted by SELKER
and YANOFSKY
(1979).
This structure has a calculated AG values of -7 kcal/mol and, in the absence
of trpE translation, could partially block base pairing with the 3’ end of 16s
ribosomal RNA. We see no other striking regions of secondary structure, but
the nucleotide sequence before the last 100 base pairs of trpE is not yet known.
Disruption of base pairing is not, however, the only means by which ribosomes
could alter mRNA structure. Certainly, tertiary interactions could play an
important role in mRNA structure as in tRNA structure (RICH and RAJBHANDARY 1976). Very little is presently known about the three-dimensional requirements for an efficient translation start site.
Examination of the nucleotide sequence in the regions between the two pairs
of cistrons under consideration reveals an interesting difference. The trpE
termination codon overlaps the trpD initiation codon by one base:
-
t r p E -T h r - P h e -en d
~
ACU UUC UGAUG
_ _ -GCUMet-Ala-trpD
(NICOLS
et al., in preparation). The start and stop codons in the trpC-trpB inter-
cistronic region, on the other hand, are separated by 11 bases (CHRISTIEand
PLATT,
in preparation; see also SELKER
and YANOFSKY
1979). By virtue of the
792
D. S. O P P E N H E I M A N D C. YANOFSKY
overlapping stop and start codons, a ribosome terminating translation of trpE
mRNA would immediately be placed in an initiating environment on trpD
mRNA. The overlapping punctuation could thus provide a mechanism to insure
that the same ribosome (or a component thereof) translates these two contiguous
genes. Such a mechanism would presumably not operate between trpC and trpB
with their 11 base-pair intercistronic sequence.
MARTIN
and WEBSTER
(1975) studied the fate of ribosomal subunits at the
translation termination signal of the coat protein gene of the RNA bacteriophage
f2. They showed that while the 50s ribosomal subunit rapidly dissociates at the
termination signal, the 30s subunit remains transiently attached to the RNA, its
rate of dissociation varying with the in uitro conditions. They concluded that the
ability of a terminated 30s subunit to reinitiate depends on, among other factors,
the proximity of the next initiation region. Thus, it is possible that the 30s subunit of a ribosome terminating trpE mRNA translation could immediately initiate trpD mRNA translation, since it will already be bound at the trpD initiation
site.
ZALKIN,
YANOFSKY
and SQUIRES (1974) showed that ribosomes that translate
trpE mRNA do not translate trpD mRNA in the presence of kasugamycin, a specific inhibitor of translation initiation. From this, it was concluded that ribosomes
must discharge and reattach between trpE and trpD mRNA translation. However, kasugamycin is known to inhibit f-Met-tRNA binding to RNA-attached
70s ribosomes (OKUYAMAet al. 1971). Thus, the kasugamycin sensitivity of trpD
mRNA translation may be consistent with nondissociation of ribosomes at the
trpE-trpD intercistronic boundary. PETERSEN
et al. (1978) have presented data
consistent with nondissociation of ribosomes between cistrons during translation
of lac mRNA.
One further difference between the two pairs of genes under consideration is
in the fate of their polypeptide products. The trpE and trpD polypeptides are
found in equimolar amounts in a tetrameric functional enzyme complex in the
cell (YANOFSKY
1971). The trpC and trpB gene products on the other hand do
not form a complex with each other.
The efficiencyof suppression of nonsense suppressors (GAREN1968; YAHATA,
OCADAand TSUGITA
1970) and polarity suppressors (A. WESSELING
and I. CRAWFORD, personal communication; this study, see Tables 1, 3, 4) varies with the
particular mutation being suppressed. Therefore, the kind of analysis presented
in this study requires the presence of a gene downstream from the pair of genes
being examined for translational coupling. The expression of the downstream
gene serves as an independent measure of the efficiency of the introduced suppressors. Thus, in the absence of a downstream gene for comparison, an analysis
of trpB polar mutations like our analysis of trpE polar mutations is not possible.
Although the existence of translational coupling between trpB and t r p A has not
been assessed, several points bear mentioning. The intercistronic nucleotide
TRANSLATIONAL COUPLING
793
sequence between these two genes has the same form as that between trpE and
trpD :
t r p B ---G l u - I 1e - e n d
GAA AUC UGAUG
GAA__M e t - G 1u - t r p A
(PUTT and YANOFSKY1975). Furthermore, as in the case of trpE and trpD,
the trpB and t r p A polypeptides form an equimolar functional complex in the
cell (YANOFSKY
and CRAWFORD1972). Finally, just as the weakest trpE polar
mutant is still quite polar on trpD, no trpB mutation that is only weakly polar
on t r p A expression has been isolated. Even the most distal trpB polar mutation
and I. CRAWFORD,
personal
allows only 30% of t r p A expression (A. WESSLING
communication), whereas the most distal trpD and trcC chain-termination mutations are nonpolar (YANOFSKY
et al. 1971). It is conceivable, therefore, that
translational coupling between trpB and t r p A similar to that we observe between
trpE and trpD could be demonstrated. For example, the same kind of analysis
as performed here could be applied to trpB polar mutations in a genetic background in which another gene had been inserted between the end of t r p A and
the trp operon transcription termination signal.
The nucleotide sequence of the trpD-trpC intercistronic region is as yet undetermined; hence, it cannot be correlated with the small translational coupling
effectobserved between these two genes.
Perhaps the coupling that we observe between translation of the mRNA segments corresponding to the first two genes of the trp operon represents a means
to insure equimolar production of polypeptides that function as a complex in the
cell. The overlapping stop and start codons punctuating such pairs of genes could
reflect the mechanism for such coupling (SELKERand YANOPSKY
1979).
The authors are indebted to VIRGINIAHORNand MIRIAMBONNER
for their assistance. These
studies were supported by grants from the Public Health Service (GM-09738) and the National
Science Foundation (PCM 77-24333). DANIELS. OPPENHEIMwas a predoctoral fellow of the
National Science Foundation. CHARLESYANOFSKY
i s a Career Investigator of the American
Heart Association.
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Corresponding editor: D. SCHLESSINGER