J. Mol. Biol. (t990) 216, 25-37
Replacement of the Escherichia coli trp Operon Attenuation
Control Codons Alters Operon Expression
Robert Landick~'21-, Charles Yanofsky 3, Karen C h o o ~ and Le P h u n g ~
Departments of 1Biology and 2Biochemistry and Molecular Biophysics
Washington University
St Louis, MO 63130, U.S.A.
and
SDepartment of Biological Sciences
Stanford University
Stanford, CA 94305, U.S.A.
(Received 5 February 1990; accepted 8 June 1990)
To test features of the current model of transcription attenuation in amino acid biosynthetic
operons, alterations were introduced into the trp operon leader region and expression of the
mutated operons was examined in miaA and miaA + Escherichia coli strains that lacked the
trp repressor. The miaA mutation prevents modification of the adenosine residue
immediately 3' of the anticodon of tRNAs that interact with codons beginning with uridine.
The undermodified tRNA Trp in miaA strains is thought to increase readthrough at the trp
attenuator by slowing ribosome movement over two tandem Trp codons in the 14-codon
leader peptide coding region. The rate of translation of these two "control codons" is
thought to be the key step in determining the extent of transcription attenuation in the trp
leader region. Sequential deletion of trpL DNA specifying the leader peptide initiation
region, RNA segment l, RNA segment 2 and RNA segment 3 alternately decreased and
increased trp operon expression, a result consistent with previous findings in another
bacterium and the generally accepted model for transcription attenuation. Replacement of
the tandem Trp control codons by AGG-UGC (Arg-Cys) codons eliminated the miaAdependent increase in transcription readthrough. Replacement of the Trp control codons by
AGG-UGA (Arg-stop) codons caused complete reaxtthrough at the trp attenuator as well as
abolisi~'mg the miaA effect. Presumably, the ribosome terminating translation at the new
UGA c4don mimics the effect of a stalled ribosome at the Trp control codons. This finding
suggests that ribosome dissociation at some stop codons is slow relative to the time required
for transcription of the trp leader region. Thus, most ribosomes translating the trp leader
peptide coding region may remain attached to the natural UGA stop codon until after the
attenuation decision is made. This interpretation supports models for trp operon attenuation
in which the elevated basal level readthrough is determined by occasional ribosome release
prior to synthesis of the 3 : 4 terminator hairpin.
1. I n t r o d u c t i o n
these mechanisms is that for the trp operon of
Escherichia coli, where experimental evidence has
been provided in support of many of the features of
the attenuation model (Landick & Yanofsky, 1987;
Roesser & Yanofsky, 1988; Roesser et al., 1989;
Landick et al., 1985, 1987; Kolter & Yanofsky,
1984). As currently postulated, the first distinguishable step following transcription initiation is the
formation of a paused transcription complex, triggered by formation of the 1:2 RNA hairpin after
synthesis of the first 92 nucleotides of the transcript
(Farnham & Platt, 1981; Winkler & Yanofsky,
Many amino acid biosynthetic operons in enterobacteria are regulated by transcription attenuation
mechanisms that are similar in their key features
(Landick & Yanofsky, 1987; Bauer et al., 1983;
Kolter & Yanofsky, 1982). The best understood of
¢ Author to whom all correspon.dence should be
addressed.
:~ Present address: Department of Biochemistry and
Molecular Biology, The University of Chicago, Chicago,
IL 60637, U.S.A.
0o22-2836/90/200025-13 $03.00/0
25
© 1990 Academic Press Limited
26
R. Landick et al.
1981; Fisher & Yanofsky, 1983; Fisher et al., 1985;
Landick & Yanofsky, 1984). This transcription
pause presumably provides time for a ribosome to
initiate translation of the trp leader peptide coding
region. Translation of the 5'-proximal portion of the
leader peptide coding region releases the paused
transcription complex, perhaps because the moving
ribosome pulls RNA segment 1 into its mRNA
binding site, dissociating the 1:2hairpin, and
allowing re-formation of a normal RNA:DNA
heteroduplex transcription substrate (Landick et al.,
1985). The translating ribosome then continues
translation until it either reaches the UGA stop
codon or, if there is a deficiency of charged tRNA Trp,
stalls at the Trp control codons. When the latter
occurs RNA segment 1 is covered by the ribosome,
RNA structure 2:3, the antiterminator, forms and
precludes formation of RNA structure 3:4, the
terminator. Thus, the consequence of ribosome
stalling at the Trp control codons is transcription
through the attenuator into the trp operon structural genes.
One of the few remaining features of this mechanism requiring experimental clarification is the dynamics of ribosome movement and release relative to
RNA polymerase movement (Kolter & Yanofsky,
1984; Roesser & Yanofsky, 1988; Roesser et al.,
1989). The absence of leader peptide synthesis in a
trpL29t (leader peptide AUG-~AUA) strain
decreases trp operon expression by a factor of 2 to 3
relative to the basal level observed in wild-type
strains growing in excess Trp (Kolter & Yanofsky,
1984; Zurawski et al., 1978; Roesser et al., 1989).
Reduction of basal level readthrough when leader
peptide synthesis is prevented has been termed
superattenuation (Stroynowski & Yanofsky, 1982;
Yanofsky, 1984). It also has been documented in
Serratia marcescens, where deletion o f the leader
peptide Shine-Dalgarno sequence or AUG codon
decreases trp operon expression by a factor of 7 to
l0 (Stroynowski//et al., 1982; Stroynowski &
Yanofsky, 1982).~Roesser & Yanofsky have shown
that wild-type ba'sal level readthrough may be attributed to ribosome release from the leader peptide
stop codon, since inhibition of release in release
factor mutants decreases trp operon expression
(Roesser & Yanofsky, 1988; Roesser et al., 1989).
They have argued that the 15°/o basal level readthrough observed in wild-type strains growing in
excess Trp can be explained by approximately 24 %
of the ribosomes releasing from the leader peptide
UGA codon prior to 3 : 4 terminator RNA synthesis,
t trpL is the traditional genetic designation for the
trp leader region; that is, the transcribed portion of the
trp operon that precedes the trpE AUG codon, trpLep is
our designation for the altered trp leader region
containing EcoRI and PstI sites (see Figs 1 and 2).
Alterations to trpLep are designated by the position of
the alteration and the nucleotide present in the
derivative, or by A when the nucleotide has been
deleted. Deletions of larger portions of trpLep are
numbered.
with half of the released RNAs folding into the
antitermination configuration (Roesser et al., 1989).
To characterize further the fate of a ribosome
that translates the trp leader peptide coding region,
as well as test general features of the current model
for attenuation, we constructed several altered trp
leader regions and examined transcription of these
variants both in vivo and in vitro. We constructed
several deletions of the trp leader region that
blocked synthesis of the leader peptide and removed
increasing numbers of RNA secondary structurespecifying segments. Examination of these deletions
supported the postulated role in attenuation of
these elements and also provided controls for a
second set of experiments. Here we tested replacements, either with AGG-UGC (Arg-Cys) or
AGG-UGA (Arg-stop) codons, of the Trp control
codons in the trp leader peptide coding region.
Results from these experiments support the idea
that a ribosome that reaches a stop codon releases
slowly relative to the rate of transcription of the trp
operon leader region.
2. Materials and Methods
(a) Bacterial strains, plasmids, media and
growth conditions
The bacterial strains and plasmids used in this study
and their derivations are listed in Table 1. Transfer of trp
leader regions from plasmids to the chromosomes of RK2
and RK3 was accomplished by recombination essentially
as described by Kolter & Yanofsky (1984). All t~T leader
DNAs were transduced from the strain in which recombination occurred into plasmid-free RK2 and RK3, using a
generalized P1 transducing phage and selecting for
growth in the absence of tryptophan, to ensure that the
strains used for assay of trp operon expression were
isogenic except for the desired trpL alterations. Difco
Tryptic Soy Broth was prepared according to the manufacturer's instructions. Luria-Bertani (LB) broth (Miller,
1972), Terrific Broth (Tartof & Hobbs, 1987) and
Vogel-Bonner medium (Vogel & Bonner, 1956) were
prepared as described.
(b) Chemicals and enzymes
High pressure liquid chromatography grade ethyl
acetate (Baker) and scintillation grade Triton X-100
(Packard) were used for anthranilate synthase assays. All
other chemicals were from Sigma (St Louis, MO).
Restriction endonucleases and DNA-modifying enzymes
were obtained from New England Biolabs (Beverley, MA),
Promega Biotech (Madison, WI) and IBI (New Haven,
CT) and used according to the manufacturers' instructions. E. coti RNA polymerase was prepared as described
by Burgess & Jendrisak (1975) or was a gift from
M. Chamberlin (University of California, Berkeley). NusA
protein was purified as described by Schmidt &
Chamberlin (1984) from an overproducing E. coli strain
(Gribskov & Burgess, 1983). Oligodeoxyribonucleotides
were synthesized on an Applied Biosystems model 380B
oligonucleotide synthesizer, either at Stanford University
or at Washington University, using standard phosphoramidite chemistry.
Replacement of trp Operon Control Codons
27
Table 1
E. coli strains and plasmids
Strain or
plasmid
Genotype or derivation
Reference
A. 81rains
RL245
RL246
RL289
RL290
I~L291
RL292
RL293
RL294
RL295
RL296
RL297
RL298
RL299
RIA39
RL440
RL441
RIA42
RL443
RL444
RIA45
supE thi A(lac-proAB)/F' traD36 proAB +
laclqZAM15
recA1 endA1 gyrA96 thi hsdRl7 (rk-, mk+)
~tpE44 relA1 A(lac-proAB)/F' traD36
proAB + laclqZAM15
W3110 AtrpL-11/180 tnaA2 trpR
W3110 AtrpL-ll/180 tnaA2 trpR miaA
W31 l0 tnaA2 trpR
W31 l0 tnaA2 trpR miaA
W3110 trpLep tnaA2 trpR
W3110 trpLep tnaA2 trpR miaA
W3110 trpLep50A,57A,62C tnaA2 trpR
W3110 trpLepSOA,57A,62C tnaA2 trpR miaA
W3110 trpLep57A,62A lnaA2 trpR
W3110 trpLep57A,62A tnaA2 trpR miaA
W3110 trpLep57A,62C tnaA2 trpR
W31 l0 trpLep57A,62C tnaA2 trpR miaA
W31 l0 trpLep50A,57A62C,63G tnaA2 trpR
W3110 trpLepSOA,57A.62C,63G tnaA2 trpR
miaA
W3110 trpLepASO.57A,62C tnaA2 trpR
W3110 trpLepA50,57A,62C tnaA2 trpR miaA
W3110 AtrpLeplO1 tnaA2 trpR
W3110 AtrpLepl01 tnaA2 trpR miaA
W3110 AtrpLepl02 tnaA2 trpR
W31 l0 AtrpLepl02 tnaA2 trpR miaA
W3110 AtrpLepl03 tnaA2 trpR
W31 I0 AtrpLepl03 tnaA2 trpR miaA
W3110 AtrpLepl04 tnaA2 trpR
W3110 AtrpLepl04 tnaA2 trpR miaA
W3110 AtrpLepl05 tnaA2 trpR
W3110 AtrpLepl05 tnaA2 trpR miaA
W3110 AtrpLep106 tnaA2 lrpR
W31 l0 AtrpLepl06 t~mA2 trpR miaA
W31 l0 AtrpLepl07 tnaA2 trpR
W31 l0 AtrpLepl07 tnaA2 trpR miaA
W3110 AtrpLepl08 tnaA2 trpR
W3110 AtrpLepl08 tnaA2 trpR miaA
W3110 AtrpLeplOltrpLep57A,62A tnaA2 trpR
W31 l0 AtrpLep101trpLep57A,62A tnaA2 trpR
R~446
RL447
miaAAtrpLeplO1trpLep57A,62C tnaA2 trpR
W3110
W3110 AtrpLep101trpLep57A,62C tnaA2 trpR
RIA48
RL449
W3110 At.rpLep105trpLep57A,62A tnaA2 trpR
W3110 AtrpLeplO5trpLep57A,62A tnaA2 trpR
RIA50
RL451
W31 l0 AtrpLeplO5trpLep57A,62C tnaA2 trpR
W3110 AtrpLeplO5trpLep57A,62C tnaA2 trpR
JM101
JM 109
RK2
RK3
RL224
RL225
RL230
RL231
RL233
RL234
RL236
RL237
RL239
RL240
RL242
RL243
Yaniseh-Perron eta/. (1985)
Yanisch-Perron et al. (1985)
Kolter & Yanofsky (1984)
Kolter & Yanofsky (1984)
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miaA
B. Plasmids
pRL243
pRL410
pRL415
pRIA16
pRL419
pRIA20
pRIA21
pRIA22
pRIA23
Wild-type trp 650 HpaII fragment cloned in
pUCll AccI site
trpLep 490 Sau3A fragment in BamHI site of
pUCll9 with trp and lac promoters in the
same orientation
trpLep SmaI-SalI fragment from pRIA10
inserted between the EcoRI and HindIII sites
of pUC119 using DNA polymerase Klenow
fragment to fill-in 5' overhangs, trp and/ac
promoters in opposite orientations
Same as pRIA15 with trp and lac promoters in
the same orientation
pRIAI6 containing trpLep50A,57A,62C
pRL416,contabfing trpLep57A,62A
pRIAI5 containing trpLep57A,62C
pRIA15 containing trpLep50A,57A,62C,63G
pRIA15 containing trpLepA50,57A,62C
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R. Landick et al.
28
Table 1 (continued)
Strain or
plasmid
)RL441
)RL442
)RL443
)RL444
)RIA45
)RIA46
)RL447
)RL448
)RL449
)RL450
)RIA51
)RIA52
Genotype or derivation
Reference
p R L 4 1 5containing AtrpLepl01
pgL415 containing AtrpLep102
pRIA15 containing AtrpLepl03
pRIA15 containing AtrpLepl04
pRL415containing AtrpLeplO5
pRIAl5 containing AtrpLepl06
pRIA15 containing AtrpLepl07
pRIAl5 containing AtrpLepl08
pRL415containing AtrpLep101trpLep57A,62A
pRIAl5 containing AtrpLep101trpLep57A.62C
pRIA15 containing AtrpLep105trpLep57A.62A
pRIAI5 containing AtrpLeplOStrpLep57A,62C
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t Constructed by reconibination of RK2 with the appropriate plasmid listed and selection for Trp +
followed by Plvir-mediated transduetion of the mutant trp allele into RK2.
Constructed by recombination of RK2 with the appropriate plasmid listed and selection for Trp +
followed by Plvir-mediated transduction of the mutant trp allele into RK3.
(c) D N A isolation, preparation and analysis
Plasmid DNAs were prepared on a small scale using the
alkaline lysis procedure (Maniatis et al., 1982) and on a
large scale using the Triton X-100 lysis procedure
(Silhavy el al., 1984) followed by CsCI/ethidium bromide
buoyant density gradient centrifugation (Maniatis el al.,
1982). DNA fragments for in vitro transcription experiments were isolated by non-denaturing polyaczTlamide
gel electrophoresis of appropriate plasmids that had been
digested with XbaI and BamHI, or subjected to the
polymerase chain reaction (Higuchi el al., 1988) with
appropriate oligonucleotide primers, followed by elution
with X buffer (Maxam & Gilbert, L980). Unless otherwise
stated, all DNA manipulations were performed following
standard published protocols (Ausubel el al., 1989;
Maniatis et al., 1982). DNA sequences were determined by
the dideoxynucleotide sequencing method (Ausubel el al.,
1989; Sanger el al., 1977) using phage T7 DNA polymerase
(Sequenase ~, US Biochemieals), [a-asS]dATP (Amersham)
and either unive/rsal or trp-specific oligonucleotide
primers.
(d) Construction of pRL415 containing the trpLep
reaction was terminated by thorough mixing with 8 pl of
250mM-Na2EDTA, (pH 8"0) and 25pl of equilibrated
phenol. After recovery and extraction of the aqueous
layer with CHCI3, the partially double-stranded, linearized DNA was electrophoresed through an 0"80/0 (w/v)
low-melting agarose (IBI)/TBE gel and isolated by phenol
extraction of a gel slice. After recovery by precipitation
with ethanol, the DNA was ligated to a phosphorylated,
synthetic duplex DNA fragment [d(ACTGCAGAAAGGTTGGTGGCGCACTTCCTGAATTCGGGC)/d(CCCG AATTCAGGAAGTGCGCCACCAACCTTTCTGCAGT)]
and
transformed into strain JMl01. A recombinant phage
containing the desired sequence was identified by restriction endonuclease site analysis and DNA sequencing. The
trpLep leader DNA then was excised from the recombinant Ml3 phage with S~mI and SalI, made blunt-ended
by treatment with DNA polymerase I Klenow fl'agment
and ligated to pUCll9 (Vieira & Messing, 1987), which
had been cleaved with EcoRI and HindIII and similarly
made blunt-ended, so that the EcoRI and PslI sites in the
trpLep DNA were unique in the resulting plasmid
(pRIAl5).
leader region
(e) Construction of trp leader deletions'
The trpLep DNA was constructed using a wild-type
lrpL 490 bp~ ,.~au3A DNA fragment inserted into the
BamHI site of M13 phage mp9 (Messing & Vieira, 1982).
pRL415 that had been cleaved with PstI was treated
with Bat31 nuclease (New England Biolabs) for 30 s at
37°C, repaired with DNA polymerase I Klenow fragment
and ligated to EcoRI linkers [d(CGGAATTCCG)]. After
removal of excess linkers by digestion with EcoRI and
spermine precipitation, the DNA was recircularized with
DNA ligase and transformed into strain JM109. The
extent of deletion in plasmids recovered was determined
by restriction endonuclease site mapping and subsequent
DNA sequencing. The deletions shown in Fig. 2 were then
assembled by religation of the appropriate DNA fragments excised from low-melting agarose gels after generation, by cleavage, of different deletion plasmids and
pRIA15 with EcoRI and BamHI (AlrpLepl02,
AtrpLepl03, AlrpLepl04, AlrpLepl06, AtrpLepl07 and
AlrpLepl08) or by cleavage with PslI or EcoRI followed
by generation of blunt-ends with phage T4 DNA polymerase and DNA polymerase I Klenow fragment, respectively, and then BamHI (AtrpLepl01 and AlrpLepl05).
Approx. 1/~g of single-stranded DNA from this phage was
hybridized to a 10-fold molar excess of double-stranded
490 bp Sau3A DNA fragment at 100°C for 90 s followed
by 60 min at 68°C in 92/~l of l0 mM-Tris" HCI, (pH 7"4),
l0 mM-MgCl2, 6 mM-KCI, 100 ~g of BSA/ml. After addition of fl-mercaptoethanol to 6 mM, the DNA was digested
with 20 units of HphI (New England Biolabs) for 20 min
at 37°C, combined with 4/d of 5mM-Trp, 5~1 of
1 M-NaCI, l ]~g of trp repressor (to prevent cleavage at the
RsaI site in the trp operator), and digested with l0 units
of RsaI (New England Biolabs) for 15 min at 37°C. The
Abbreviations used: bp, base-pair(s); BSA, bovine
serum albumin; DTT, dithiothreitol.
Replacement of trp Operon Control Codons
(f) Construction of trp leader regions with altered
control codons
Approx. l ttg of pRIA15 was cleaved with EcoRI and
PstI and the large DNA fragment was recovered in a slice
of low-melting agarose after electrophoresis. Approx. 1/50
of the gel slice was melted and combined with approx.
5 ng of the phosphorylated, sinffle-stranded oligonucleotide
[d(AATTCAGGAAGTGCGTCACCTACCTTTCTGCA)]
(where the duplicated letters indicate incorporation of
50~/o of each base at that position), ligated and transformed into JM109. The altered trp leader DNAs shown in
Fig. 2 were identified by screening of plasmids from the
transformants for loss of the HhaI site and acquisition of
an NlaIV site followed by DNA sequencing.
(g) In vitro transcriplion assays
For synchronized, single-round transcription reactions
to measure paused transcription complex half-lives,
2"5 p,nol of RNA polymerase and 1 pmol of DNA
template were combined and incubated at 37 °C for l0 min
in 100/~l of buffer containing 20 mM-Tris-acetate,
(pH 8-0), 130 mM-KC1, 4 mM-MgCI2, 0-1 mM-Na2EDTA,
0"1 mM-DTT, 150pM-ATP, 20gM-[~-32p]GTP, 4% (v/v)
glycerol. 20/~g of acetylated BSA/ml. Transcription was
initiated by addition of (.'TP and UTP (150 #M each, final
concentration) and l0 pg of rifampicin/ml. Samples (10 pl)
were removed at appropriate time intervals and mixed
with l0 pl of 2 x TBE, 0"1% (w/v) bromphenol blue, 0"1%
(w/v) xylene cyanol saturated with urea. RNA samples
were analyzed by electrophoresis through a 10% (w/v)
polyacrylamide/7 M-urea-TBE gel. The radioactive pause
RNA bands were excised from the gel and their C,erenkov
radiation was determined in a scintillation counter. After
subtraction of appropriate background values, these data
were plotted versus time on semi-log paper and the pseudo
first-order rate of pause RNA disappearance was determined from the slopes of the semi-log plots.
Steady-state transcription reactions to determine percentage readthrough at the trp attenuator were performed
under the same conditions, but with DNA templates
prepared from pl~hmids and forward and reverse pUCll9
sequencing primers by the polymerase chain reaction
(Higuchi et al., 1988). After formation of open complexes
for l0 min at 37°C, heparin was added to 10#g/ml, and
CTP and UTP to 150#M, and the reactions were incubated for 10min at .37°C. After electrophoresis as
described above, the R,NAs were quantitated in the gels
using an AMBIS v Radioanalytic Imaging System.
Percentage readthrough was calculated as: mol ~o run-off
RNA/(mol % leader RNA +mol ~o run-off RNA).
{h) Secondary structure prediction
rCNA secondary structures and their free energies of
stabilization were predicted using the RNAFLD program
of Zuker & Steigler {1981) as implemented by the
University of Wisconsin Genetics Computer Group on a
VAX 11/750 in the Department of Biology, Washington
University.
(i) Anthranilate synthase assays
Anthranilate synthase assays <vere performed essentially as described by Creighton & Yanofsky (1970}. Cells
for anthranilate synthase assays were grown in Tryptic
Soy Broth at 37°C to saturation. The cells were diluted
29
1 : 100 into 5 ml of the same media in 15 mm x 125 mm
culture tubes and grown at 37°C on a New Brunswick
Scientific Rollodrum apparatus at 60 revs/min. Growth
was monitored in a Klett-Summerson Colorimeter and
halted by placing the cells on ice when the density
reached approx. 80 to 100Klett units (660filter). A
volume equal to 400 Klett units was transferred to a
15ml corex tube and the cells were collected at
10,000 revs/min for 5 rain in a Sorvall SA-600 rotor. After
washing once with cold l x Vogel-Bonner salts (Vogel &
Bonnet, 1956), the cells were permeabilized by suspension
in 200gl of 100mM-Tris-HCl, (pH 7-8), 0"1% Triton
X-100, freezing in a dry ice/ethanol bath, and thawing on
ice. Five gl of cell suspension was mixed with 500 tti of
100 mM-Tris- HCI, (pH 7"8), 5 mM-Mg(CH3CO0=)2, 5 mML-glutamine, 1 mM-DTT, 150 ttg of barium chorismate/ml
and incubated at 37°C with shaking for 20 min. The
reactions were stopped by placing in an ice-water slurry
and adding 100ttl of 1 M-ammonium acetate, (pH 4"5).
Anthranilic acid was extracted into 3 ml of ethyl acetate
and quantitated by fluorescence in an Aminco-Bowman
spectrofluorimeter
(excitation = 340 nm,
emission
: 4 0 0 nm).
3. R e s u l t s
(a) The altered region, trpLep, does not affect
regulation of trp operon attenuation
We constructed a variant trp leader region t h a t
contains a PstI restriction endonuclease recognition
site, an e x t r a Gln codon (CAG) at the base of
segment 1 and an EcoFCI site just before the stop
codon in the loop of the 1 : 2 R N A hairpin region
(Figs 1(c) and 2; see Materials and Methods). These
changes had a negligible effect on the theoretical
stability of the 1 : 2 RNA hairpin (Table2). To
verify t h a t the changes in the trpL region did not
substantially alter transcription pausing and termination in the absence of translation, we performed
in vitro transcription analyses with DNA fragments
bearing the alterations, using both steady-state and
single-round, synchronized protocols (see Materials
and Methods). The half-life of trpLep paused transcription complexes was essentially identical to t h a t
of the wild-type trpL paused transcription
complexes, in both the absence (Table 2) and presence of NusA protein (approx. 3-fold increase in
pause half-life; d a t a not shown). Further, the percentages of transcription complexes t h a t read
through the trpLep and wild-type trp a t t e n u a t o r s in
vitro were nearly identical (Table2). Thus, the
alterations of the trpL sequence introduced b y
incorporating the PstI and EcoRI sites did not
affect the transcriptional properties of the leader
region. We also compared the levels of anthranitate
synthase produced in trpLep and trpLep miaA
strains grown in a rich medium to those of isogenic
wild-type trp strains (Table 3). The trpLep strains
gave levels essentially identical to the wild-type,
confirming t h a t the nucleotide changes in trpLep do
not alter regulation of trp operon expression. Similar
results were obtained by Roesser & Yanofsky using
independently derived strains grown in a minimal
R. Landick et al.
30
1:2
A
A
2:3
A
U
A
G
A
C-- G too
G--C
A
Stop 70 G
C
G
C------G
C------G
U
C
U=A
C----Gso
A=U
C----G
G
U.
U
AAGUUCACG
U
A
A
A
A
A
G
G
G
Set
Thr
AU=AA
C
U
CAc_GAC
3:4
AAU
U
G
Arg
A
C
P~
soC
t2O C=_G
G
UU
G--C
U
Trp
G=--C
C-~G
A
U=A
C~G
U
Trp G_=C
C--G
AC
Met LiPs A/a lie Phe Vat Leu Lys Gly G -~=c so
G -~-C
GACAAUGAAAGCAAUUUUCGUACUGAAAGGUU = A
AUCAGAGACCCA = U UUUUUUU
30
40
so
(o )
U=A
PRUSE
140
U---A
UA=U
U----A
ao GuG--C%
A C--=GA
G--C
G--C
Trp An3 Thr Set Stop G----C
UGGUGGCGCACUUCCUGAAA C -= GCCUAA
Trp
60
,,#'G--= C
C--G~oo
G
120
(b)
A
UAA
EcoRI
EcoRI
AU
AU
,op
0
EcoRI
AU
G2f-Y,C
stop GV - ' f , C
U
G
C=G 8o
Set' ;'o C==G
U
C
U:A
Thr
C==G
A--U
C=G
,~ G
uA
C
U
u
G
C_=G so
Ser 70 C--=G
U
C
U:A
C--=G
Thr
A=U
C~-G
~
G
uA
C
G
C--G e~
7o C-~G
U
C
U=A
C--G
A=U
C=-G
G
uA
~=uUSO
G--=C
~o O.---h
Trp G---C
tie Phe Val Leu Gin Lys GJ), G ~ C
PRUSE
AUUUUCGUACUGCAG/C~AGGUU=A
~"
40
Psi!
so
U
6o U = A
Ar¢
G=--C
lie Phe Val Leu Gin Lys Gty G ~ C
PRUSE
AUUUUCGUACUGCAGA,~GGUA
A p/
40
PStl
(C)
~"
(d)
U
6o U = A
,4ql
fi=C
lie Phe Val Leu Gin LyS Gly G -=C
pRmmsE
AUUUUCGUACUGCAGAAAGGUA
A ~
4o
PSf t
so
U
(e)
Figure 1. Alternative secondary structures in the trp leader RNA. (a) Wild-type trp terminated leader RNA (complete
terminated transcript) folded into structures 1:2 and 3:4 in the configuration thought to produce transcription
termination. (b) Antiterminator, the alternative 2 : 3 RNA folding pattern for the wild-type leader transcript thought to
produce transcription readthrough. (c) The 1:2 pause RNA hairpin of trpLep. (d) The 1:2 pause RNA hairpin of
trpLep57A,62C. (e) The 1:2 pause RNA hairpin oflrpLep57A,62A. Nucleotide changes in various mutants are indicated
by bold type. Secondary structure predictions are based on the algorithm of Zuker & Steigler (1981), disallowing
potential G" U base-pairs at the ends of paired regions.
medium (Roesser & Yanofsky, 1988; Roesser et al.,
1989).
(b) Sequential deletion of the leader peptide
initiation region, RNA segment 1, RNA
segment 2 and RNA segment 3 alternatively
decreased and increased readthrough
of the trp attenuator
We constructed a series of deletions t h a t removed
increasing lengths of trpL, starting near the transcription start site (see Materials and Methods). Two
sets of deletions were engineered, one beginning at
+ 9 of trpL and a second at +15. Each set
contained four members in which the 3' end of the
deletions extended to +49, +72, +112 and +134
of trpLep, respectively, and carried an EcoRI linker
in place of the deleted region (Fig. 2). We tested
each deletion for its ability to direct transcription
termination in an in vitro transcription system using
purified RNA polymerase and found t h a t removal
of segment 1 or segments 1, 2 and 3 almost eliminated termination, whereas removal of only the
leader peptide initiation region or segments l and 2
permitted termination (Fig. 3, Table 2). These
effects were mirrored in an analysis of anthranilate
synthase levels in strains bearing the various deletions (Table 3). All of the deletions eliminated the
effect of the miaA mutation on anthraniliate
synthase levels, consistent with the idea t h a t miaA
defects exert their effect on attenuation by slowing
ribosome translation of Trp codons, since none of
the deletions contains a leader peptide coding region
(Fig. 2, Table 3).
The effects of the deletions in vivo were less than
what might be postulated from the model of
attenuation and the reported 15~/o attenuator readthrough in wild-type strains growing in excess Trp.
For example, AtrpLepl02, AtrpLepl04, AtrpLepl06
and AtrpLepl08 gave only a threefold increase in
anthranilate synthase rather than the 6"5-fold
increase t h a t would be predicted if there was a
1
MetLysAlaIlePheValLeuGlnLysGlyArgCysArgThrSerStop
3
4
GCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGC
100
|10
120
130
T T TTT TT T
140
CGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
80
90
! O0
! lO
!20
! 3,0
140
2
MetLysAla I lePheVa iLeuGlnLysGlyTrpTrpArgThrSer St op
AAGT TCACGTAAAAAGGGTATCGACAATGAAAGCAATTT T CGTACTGCAGAAAGGT TGGTGGCGCAC TTCCTGAAI~CGGGCAGTGTATTCACCAT
I0
20
30
40
50
60
70
80
90
LysG lyT r pT r pArgTh rSe r'5top
AAAGGTTGGTGGCGCACTTCCTGAAA
50
60
70
AAGTTCACCGGAATTCCG
AAGTTCACCGGAATTCCG
z~trpLep107
~trpLepl 08
GAAAGGT~GGTGCCGCACTTCCTGAATTCGGGCAGTGTAT
TCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCT
TTTTTTT
GAAAGGT~GGTG~CG~ACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
GAAAGGT~GGTG~CGCACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATA~c~AGCCCGCCTAATGAG~GGGCTTTTTTTT
GAAAGGT~GGTG~CG~ACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCG~CTAATGAGCGGGCTTTTTTTT
GGCTTTTTTTT
TACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
CGGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
GAAAGGTTGGTGGCGCACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
GGCTTTTTTTT
TACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
CGGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
GAAAGGTTGG•GGCGCACTT•CTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
F i g u r e 2. D N A s e q u e n c e s o f trp leader variants. B a s e s t h a t differ from trpLep (or from w i l d - t y p e in the case-0f trpLep) are underlined. Bases deleted in the various c o n s t r u c t s
are i n d i c a t e d b y spaces. In the AtrpLep strains, bases r e m a i n i n g from the EcoRI linkers t h a t do not correspond to trpLep sequences are underlined.
zitrpLeplOS-57A,62C AAGTTCACCGAATT
AAGTTCACCGAATT
AAGTTCAC
2arpLepl 06
AtrpLep105.57A,62A
AAGTTCACCGGAATT
z~trpLep105
AAGTTCACGTAAAACGGAATT
AAGTTCACGTAAAACGGAATTCCG
zltrpLep104
~trpLepl01-57A,62C
AAGTTCACGTAAAACGGAATTCCG
~rpLep l 03
AAGT TCACGTAAAAC GGAAT T
AAGTTCACGTAAAA
zltrpLepl 02
ZitrpLeplO1-57A,62A
AAGTTCACGTAAAACGGAATT
~rpLep101
LysVaiGlyAlaAlaLeuProGluPheGlyGlnCysIleHisHisAlaStop
AAAGGT~GGTG~GGCACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
MetLysAlaIlePheValLeuGlnLysGlyArgCysGlyThrSerStop
AAGTTCACGTAAAAAGGGTATCGACAATGAAAGCAATTTT~GTACTGCA~AAAGGT~GGTGCGGCACTTCCTGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
trpLepSOA,57A,62A,63G
MetLysAlaIlePheValLeuGln
AAGTTCACGTAAAAAGGGTATCGACAATGAAAGCAATTTTCGTACTGCA
MetLysAlal]ePheValLeuGlnLysGlyArgStop
AAGTTCACGTAAAAAGGGTATCGAcAATGAAAGCAATTTTCGTACTGCAGAAAGGT~GGTG~CGCAcTTCCTGAATTCGGGCAGTGTATTCA~cATGCGTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
trpLep50A,57A,62C
trpLepA50,57A,62C
MetLysAlaIlePheValLeuGlnLysGlyArgCysArgThrSerStop
AAGTTCACGTAAAAAGGGTATcGACAATGAAAGCAATTTTCGTACTGCA4~AAAGGT~GGTG~CGCACTTC~TGAATTCGGGCAGTGTATTCACCATGCGTAAAGCAATCAGATAcCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
trpLep57A,62A
trpLep5 7A,62C AAGTTCACGTAAAAAGGGTATCGACAATGAAAGCAATTTTCGTACTGCAGAAAGGT~GGTG~CGCA~TTCCTGAATT~GGG~AGTGTATTCA~ATG~GTAAAGCAATCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT
trpLep
Irp w ~ - ~ p @
Met LysAla I lePheVal Leu
AAGTTCACGTAAAAAGGGTATCGACAATGAAAGCAATTTTCGTACTG
l0
20
30
40
R. Landick e t al.
32
Table 2
I n v i t r o transcription pausing and termination on wild-type and altered t r p leader
DNA templates
Allele
trpL wild-type
trpLep
trpLep57A .62A
trpLep57A .62C
trpLepSOA.57A.62C
trpLep50A ,57A,62C,63G
trpLepASO.57A,62C
AlrpLeplOl
AlrpLepl02
Al~pLepl03
AtrpLepl04
AtrpLepl05
AtrpLep106
AtrpLepl07
AtrpLepl08
1:2 RNA hairpin
At7 formationt
(kcal/mol)
Paused transcription
complex half-life:~
(s)
Percentage
readthrough§
-9"9
- I 0"l
- l l'0
--7"9
- 7"9
- 7'9
- 7'9
25
26
37
21
23
17
n.d.
7'5
7"3
7"4
9"8
9"1
17
7'8
- l O"l
n.d.
14
- l 0' I
n.a.
n.a.
- 10' [
- 10'l
o.a.
n.a.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
75
7.8
93
31
82
5"0
79
t As calculated by the algorithm of Zuker & Steigler (1981), disallowing potential G-U base-pairs at
the ends of paired regions, n.a. not applicable; 1 ca1=4'184 J.
:~ Determined as described in Materials and Methods. n.d., not determined.
§ Calculated as: mol% readthrough RNAl(mol % leader RNA+mo] % readthrough RNA); see
Materials and Methods.
c o m p l e t e loss o f t e r m i n a t i o n (as was o b s e r v e d in
vitro; see Fig. 3 a n d T a b l e 2). S i m p l e d e l e t i o n o f t h e
l e a d e r p e p t i d e i n i t i a t i o n region in AtrpLeplO1 g a v e
o n l y a 25~/o d r o p in a n t h r a n i l a t e s y n t h a s e , r a t h e r
t h a n t h e 50 to 7 0 % d r o p r e p o r t e d here ( T a b l e 3)
a n d o b s e r v e d e l s e w h e r e for trpL29 ( c h a n g e o f t h e
l e a d e r p e p t i d e s t a r t c o d o n from A U G o r A U A ) . W e
do n o t k n o w i f t h e s e d e v i a t i o n s f r o m p r e d i c t i o n
resulted from altered readthrough, altered transl a t i o n o f trpE from m R N A s w i t h a l t e r e d 5' regions,
a l t e r e d m R N A s t a b i l i t i e s , oi" from t h e f o r m a t i o n o f
u n a n t i c i p a t e d R N A s t r u c t u r e s . N e v e r t h e l e s s , each
o f t h e d e l e t i o n s c h a n g e d e x p r e s s i o n o f t h e trp o p e r o n
in a m a n n e r t h a t is c o n s i s t e n t w i t h t h e c u r r e n t
m o d e l for t r a n s c r i p t i o n a t t e n u a t i o n .
(c) Replacement of the Trp control codons for the
leader peptide abolished the effect of a m i a A ,mutation
on t r p operon attenuation
T o t e s t t h e c u r r e n t l y a c c e p t e d role o f t h e T r p
c o n t r o l c o d o n s in t h e l e a d e r p e p t i d e c o d i n g region,
we a l t e r e d t h e c o n t r o l c o d o n s u s i n g trpLep while
retaining the greatest possible conservation of the
1 : 2 R N A h a i r p i n s t r u c t u r e (see M a t e r i a l s a n d
M e t h o d s ) . T w o d e s i r e d trpL v a r i a n t s were i s o l a t e d .
Table 3
Anthranilate synthase activities of various strains with altered t r p leader regions
Anthranilate synthase
activity
Relevant genotype
trp wild-type
trpLep
trpL29
AtrpLepl01
AtrpLepl05
AtrpLepl02
AtrpLep/06
AtrpLept03
AtrpLepl07
AtrpLepl04
AtrpLepl08
trp leader alteration
Trp Trp
Trp Trp
L.P. AUG--*AUA
A(L.P. AUG)
A(L.P. AUG)
A(L.P. AUG+seg. l)
A(L.P. AUG + seg. I )
A(L.P. AUG+segs 1,2)
A(L.P. AUG + segs 1,2)
A(L.P. AUG+segs 1,2,3)
A(L.P. AUG +segs 1,2,3)
miA +
miaA
(% of wild-type)t
100
100
51
83
80
280
260
120
l I0
350
270
280
280
56
81
81
300
280
81
100
330
360
t Expressed as % of the value for E. coil W3110 trpR tnaA2 (RL224) growing in Trvptic Soy Broth
+ 100/4g of Trp/mi (see Materials and Methods).
L.P., leader peptide; seg(s), segment(s).
Replacement of trp Operon Control Codons
I
2
3
4
5
6
7
8
9
- o,
Figure 3. In vitro transcription of trp leader DNAs of
various deletion mutants. Reactions were conducted as
described in Materials and Methods using DNA fragments
that had been prepared fi'om the indicated pLasmids by
the polymerase chain reaction (PCR), except for the wildtype 490 Sau3A DNA fragment (lane ! ). The bands above
the readthrough bands in the lanes using DNAs prepared
by PCR probably result from end-to-end transcription of
the amplified fragments. A fraction of these fragments
may contain ragged 3' ends that promote transcription
initiation. Bands corresponding to readthrough RNAs are
marked with upward-pointing arrows (~), whereas bands
corresponding to terminated RNAs are labeled with
downward-pointing arrows ($). Lane ], lrpL wild-type
490bp Sau3A DNA fi'agment; lane 2, pRIA41
(AtrpLepl01); lane 3, pRL442 (At~TLepl02); lane 4,
pRL443 (AtrpLepl03); lane5, pRL444 (At~TLepl04);
lane 6, pRL445 (AtrpLepl05); lane 7, pRL446
(AtrpLepl06); lane 8, pR,L447 (AtrpLepl07); lane 9,
pRL448 (AtrpLepl08).
33
One, trpLep57A,62C (Figs 1(d) and 2), contains Arg
and Cys codons in place of the Trp control codons;
the other, trpLep57A,62A (Figs l(e) and 2), contains
an Arg eodon and a stop (UGA) codon at these
positions. Both trpLep57A,62C and trpLep57A,62A
produce a trp leader transcript that lacks the potential U . A base-pair at the base of the l : 2 RNA
hairpin, although trpLep57A,62A adds a potential
A. U base-pair in the stem (Fig. 1). This reduces the
predicted stability of the trpLep57A,62C l :2 RNA
hairpin by approximately 2 kcal/mol relative to the
trpLep hairpin (Table 2). In addition to the desired
alterations, several plasmids were recovered with
other base changes between the PstI and EcoRI
sites, apparently created during repair of the singlestranded gap in the ligation product after transformation into E. coll. One of these, trp-LepSOA,
57A,62C (Fig. 2), differs from trpLep57A,62C only
by a single G to A base change in the 3'-most residue
of the PstI site. Another trpLep50A,57A,62C,63G
(Fig. 2), contains an additional C to G change at
position 63, changing the Arg codon that follows the
Trp codons to Gly. Finally, trpLepA50,57A,62C
(Fig. 2), is missing base 50, shifting the reading
frame of the trp leader peptide coding region so that
the Trp-Trp control codons are replaced by Gly-Ala,
and the position of translation termination is moved
to a UAA codon located in the loop region of the
2 : 3 RNA hairpin (Fig. l(b); wild-type positions 96
to 98).
We used these five altered trp leader plasmids to
test the importance of the Trp control codons, and
to compare ribosome step time~ versus ribosome
release and their relevance to basal level readthrough. The five trpLep control codon mutants
(Fig. 2) were also tested for in vitro pausing and
termination. Some significant variations were
detected, trpLep57A,62A produced an increased
pause half-life, consistent with the increased
stability of its 1:2 RNA hairpin. Three mutants
bearing the G to C change at trpLep position 62
(trpLep57A,62C, trpLep50A,57A,62C, and trpLep50A,57A,62C,63G) showed a reduced half-life for
the transcription pause complex (Table 2). Except
for trpLep50A,57A,62C,63G, these changes were
minimal, despite the reduction in the predicted
stability for the trpLep57A,62C 1:2 RNA hairpin
(Table 2). The reduction to 70% of wild-type level
for the trpLep50A,57A,62C,63G template is not
easily explained, but most probably reflects a reduction in the 1:2 RNA hairpin stability that is not
predicted by the aiogorithm employed for our calculations (Zuker & Steigler, 1981 ). Indeed, these calculations must be considered, at best, approximations.
The even greater increase in transcription readthrough observed with this altered template may
result from a failure of all RNA polymerase moleRibosome step time is the time required for the
ribosome to add 1 amino acid to the polypeptide chain
and translocate to the next codon on the mRNA (1 full
cycle of elongation; see e.g. Pedersen, 1984).
34
R. Landick et al.
Table 4
Anthranilate synthase activities and deduced percentage readthrough of various strains with altered trp leader
control codons
Anthranilate synthase
activity
Relevant, genotype
trp wild-type
trpLep
trpL29
trpLep57A.62A
trpLep57A .62C
trpLep50A .57A.62C
trpLepSOA.57A ,62C.63G
trpLepASO,57A.62C
AlrpLeplOl-trpLep57A,62A
AtrpLeplOl-trpLep57A,62C
trp control codons
aod leader alteration
Trp Trp
Trp Trp
L.P. AUG~AUA
Arg UGA
Arg Cys
Arg Cys
Arg Cys (Gly)
Gly Ala (frameshii't to UAA at +96)
A(L.P.AUG)-Arg UGA
A(L.P.AUG)-ArgCys
m.iaA+
miaA
(% of wild-type)~f
100
100
51
600
190
240
250
130
76
65
280
280
56
520
180
240
220
100
66
100
Deduced %
readth,'ough
miaA +
miaA
(%)~
15
15
7"fi
91
28
35
37
20
12
9"8
42
42
8"4
78
27
35
34
15
l0
15
Expressed as % of the value for E. coil W3110trpR tnaA2 (RL224)growing in TtTptic So), Broth + 100 l~gof Trl)/ml (see Materials
and Methods).
:~ Expressed as the % of RNA polymerasesthat transcribe through the trp a.ttenuatorr assuming 15% z~adthrough fi)r the wild-type
strain RL224 (Kolter & Yanofsky. 1984: Roesser el al.. 1989).
L.P., leader peptide.
cules to pause at the 1 : 2 hairpin, or increased
formation of the 2 : 3 antiterminator RNA hairpin
because 1:2 is weakened. None of the observed
changes in pausing or termination invalidate the use
of these templates in tests of the role of the T r p
codons or of ribosome release at the UGA stop
codon.
We tested control codon alterations first to deter'mine their effects on Trp control of attenuation. To
assay the effects of decreased ability to translate
Trp codons on attenuation, we recombined the
various engineered leader regions into isogenic
tnaA2 trpR miaA +/- strains (Table 1; see Materials
and Methods). The miaA mutation prevents formation of N-6-(A2-isopentenyl)-2-methylthioadenosine
and N-6-(A2-4-hydroxy-isopentenyl)-2-methylthioadenosine at the adenosine residue immediately 3' of
the anticodon of tRNAs t h a t interact with codons
beginning with U (Eisenberg et al., 1979; BjSrk,
1987). miaA was shown previously to cause a two- to
fivefold increase in trp operon expression, depending
on the growth conditions employed (Yanofsky &
Soll, 1977; Zurawski et al., 1978; Kolter & Yanofsky,
1984; Roesser & Yanofsky, 1988). In the context of
the current model for trp attenuation, this is
t h o u g h t to be caused by ribosome stalling at the
leader peptide T r p codons (despite excess Trp) due
to inefficient translation by t h e undermodified
t R N A T~p (Eisenberg et al., 1979). When the tandem
UGG (Trp) codons were replaced by an AGG (Arg)
eodon and a UGC (Cys) codon (trpLep57A,62C;
Figs 1(d) and 2), anthranilate synthase activity was
essentially the same in miaA + and miaA strains
(Table4). The same result was obtained in the
trpLep50A,57A,62C and trpLepSOA,57A,62C,63G
variants, which also lack T r p control codons
(Table 4). However, all three alterations permitted
production of higher anthranilate synthase levels
than did the wild-type or" ttTLe p leader regions in
the miaA + background (Table 4).
To determine whether this increase was due to
translation of the altered leader peptide coding
region, or to the slight destabilization of the l :2
RNA hairpin caused by sequence changes, we
constructed a trp leader region t h a t combined the
trpLep57A,62C allele with the A t r p L e p l 0 l deletion.
By removing the leader peptide initiation region,
AtrpLeplO1 should eliminate translational effects.
AtrpLeplOltrpLep57A,62C gave anthr~nilate synthase levels essentially identical to AtrpLeplOl
in both miaA + and miaA strains (Tables 3 and 4),
suggesting t h a t the increased readthrough observed
in strains bearing control codon substitutions can be
a t t r i b u t e d to effects on translation (see Discussion).
(d) A ribosome terminating translation at a UGA
codon in segment 1 prevents transcription
termination at the trp attenuator
To determine whether ribosome behavior at a
stop codon would simulate ribosome stalling at a
Trp control codon, we replaced the Trp control
codons with AGG-UGA codons (Arg-stop codons;
trpLep57A,62A; Figs l(e) and 2)). This alteration
eliminated one A. U base-pair at the bottom of the
l :2 hairpin, as did trpLep57A,62C.
Thus,
trpLep57A,62C serves as the most appropriate
control for the effect of the UGA codon at the
position normally occupied by the second T r p
control codon, Since trpLep57A,62A created one
additional A ' U base-pair and actually increased
RNA polymerase pausing at the 1 : 2 RNA hairpin
(Table 2), the consequence of rapid ribosome release
at the trpLep57A,62A UGA codon could have been
a decrease in trp operon expression relative to
trpLep57A,62C. W h a t we observed was greater
Replacement of trp Operon Control Codons
anthranilate synthase activity in trpLep57A,62A
strains (Table 4). A reasonable explanation for" these
results is that when the translating ribosome
reaches the UGA codon in trpLep57A,62A, it
remains attached to the message long enough to
prevent 1:2 formation, thereby promoting 2 : 3
antiterminator
|brmation.
We verified that
increased readthrough in trpLep57A,62A strains
was attributable to translation by testing strain
AtrpLeplOltrpLep57A ,62A.
This
strain
gave
anthranilate synthase levels nearly the same as
given by AtrpLeplO1 alone (Tables3 and 4),
showing
that
the
high
readthrough
in
trpLep57A,62A was caused by translation of trp
leader RNA.
(e) Translation over segment 2 prevents
antiterminator formation
One variant that arose in our experiments had a
single base-pair deletion at position 50 of the trpLep
leader that shifted the reading frame of the trp
leader peptide coding region so that translation
terminated at a UAA codon in the loop region of the
2 : 3 RNA hairpin (trpLepA50,57A,62C; Fig. 2). This
alteration reduced readthrough at the attenuator
slightly (Table4, compare with trpLep57A,62C).
Thus, the consequence of a ribosome translating
over segment 2 to a UAA codon where it would
block 2 : 3 tbrmation was an increase in transcription termination. This result requires some ribosomes to translate fast enough to reach segment 2
before the attenuation decision is made.
Discussion
(a) Systematic deletion of segments of E. coli trpL
confirm postulated roles for corresponding R N A
sequences in trp operon attenuation
Previous studies verified many of the basic features of the trp attenuation model by preparing and
testing various deletions of the Serratia marcescens
trp leader region (Stroynowski et al., 1982, 1983;
Stroynowski & Yanofsky, 1982). In particular, the
pattern of RNA tblding into alternate RNA secondary structures that either direct or inhibit transcription termination was established unambiguously
(see Fig. l). Base substitutions in E. coli trpL
(Kolter & Yanofsky, 1984), further reinforced the
assignment of conformations for the alternate
secondary structures. To test the role of the segments assigned to the E. coli trp leader RNA
secondary structures, we have systematically
deleted them beginning from a constant point near
the 5' end of the leader RNA. The deletions alternately decrease and increase transcription termination
at the trp attenuator both in vitro {Table 2, Fig. 3)
and in. vivo (Table 3), recapitulating in E. coli results
obtained earlier with the S. marcescens trp operon.
Deletion of the leader peptide initiation region also
provided a control for the effects of translation of
the leader peptide coding region on attenuation.
35
(b) Increased trp operon expression in strains with
rare leader peptide codons may result from slower
ribosome movement over the leader
peptide coding region
The effect on attenuation of control codon
replacements has been tested in three other amino
acid biosynthetic operons: the E. coil leu (Carter et
al., 1985) and thr operons (Lynn et al., 1987) and the
S. marcescens ilvG M E D A operon (Harms &
Umbarger, 1987). Cairo and co-workers replaced
the four Leu control codons with Thr (ACU) codons
and observed loss of regulation in response to Leu
starvation (Carter et al., 1985). However, the altered
leu operon responded only modestly to a defect in
tRNA T M charging (Carter et al., 1985). They attributed this weakened attenuation response to the
relative abundance of the tRNA T M species that
decodes ACU; the wild-type leu leader peptide
coding region contains three rare CUA codons. The
importance of the rare CUA codons, which are
decoded by a minor tRNA T M species (Ikemura,
1981), is substantiated by the decreased sensitivity
to Leu starvation of an E. coil leu operon mutant
containing CUG codons (Carter et al., 1986) and by
the ability of a single CUA codon to control
attenuation in the S. marcescens ilvGMEDA operon
(Hsu et al., 1985). Replacement of the S. marcescens
ilvGMEDA CUA (Leu) codon with a CCA (Pro)
codon completely abolishes the response to Leu
starvation (Harms & Umbarger, 1987). Finally,
Gardner and co-workers replaced the leader Thr
control codons in the E. coli thr operon with His
codons and found loss of Thr-dependent regulation
and
acquisition
of a
response
to
the
tRNAmS-modifying mutation, hisT (Lynn et al.,
1987).
We found that replacement of the tandem Trp
codons in the trp leader peptide coding region
with AGG-UGC (Arg-Cys) codons abolished
tRNAT~p-dependent regulation, but it increased
readthrough twofold in vivo (Table 4). This increase
was dependent on translation of the altered leader
peptide coding region, since deletion of the initiation region eliminated the increased readthrough
(Table 4). I n vitro, a slight increase in readthrough
was observed when trpLep57A,62C was transcribed
by purified RNA polymerase (Table 2). However,
increased readthrough in vitro was not directly correlated with readthrough in vivo, since the twofold
increase in readthrough in vitro of trpLep50A,57A,62C,63G was largely eliminated in vivo.
We conclude that translation of the leader peptide
coding region is the dominant determinant of
readthrough at the attenuator in vivo.
One explanation
for translation-dependent
increased readthrough of trpLep57A,62C is that the
presence of the rare AGG (Arg) codon decreased the
rate of translation of leader RNA, causing 15% of
the translating ribosomes to remain in the control
codon region (to account for a doubling of the 15°/o
readthrough observed at the wild-type trp attenuator; see Table 4). Decreased rates of translation of
36
R. Landick et al.
AGG codons have been observed in experiments
where they were placed in an artificial leader peptide coding region of the pyre operon (Bonekamp &
Jensen, 1988). The rare AGG codon also raises the
possibility that ribosome frameshifting might complicate interpretation of our results (Weiss et al.,
1988). Tandem AGG codons have been reported to
cause ribosome frameshifting with up to 50~/o
efficiency (Spanjaard & van Duin, 1988). However,
frameshifting at a single AGG codon has not been
demonstrated. Furthermore, a ( - ) ribosome frameshift¢ would correspond to the reading frame used
in the trpLepA50,57A,62C allele, which decreased
rather than increased levels of anthranilate synthase and a (+) ribosome frameshift is unlikely,
since it would place a UAG stop codon in the
ribosome A site (Fig. 2). The simplest explanation of
increased readthrough in trpLep57A,62C is slow
translation of the AGG codon.
(c) Replacement of a trpL Trp control codon by UGA
appears to favor antiterminator formation
Replacement o f a trpL Trp codon by UGA eliminates virtually all transcription termination in the
leader region (Table 4). The simplest explanation for
this observation is that the ribosome translating the
leader peptide coding region remains at the UGA
codon long enough to cause antiterminator formation in every transcript, thus simulating ribosome
stalling at the Trp codons in a Trp-deficient bacterium. Since the level of anthranilate synthase is
significantly higher in trpL57A,62A strains than in
strains where a Cys codon replaces the UGA, even
though the latter strains contain a less stable l :2
RNA hairpin, interpretations other than slow ribosome release at the UGA seem untenable.
This conclusion is consistent with that reached in
studies of the effect of release factor mutants on
basal level control of attenuation in the trp operon
(Roesser & Yanofsky, t988; Roesser et al., ]989).
They showed that the inhibition of ribosome release
by alterations in release factor mutants depressed
readthrough of the attenuator, presumably because
formation of the antiterminator was precluded by
the ribosome stuck at the stop codon (Roesser &
Yanofsky, 1988; Roesser et al., 1989). From an
analysis of the effect of release factor mutants on
various altered trp leaders, they argue that approximately 24~/o of ribosomes release from the wild-type
trpL UGA codon prior to complete synthesis of the
3:4 terminator RNA hairpin, and that the equal
likelihood of forming the 1 : 2 versus 2 : 3 RNA structures in the released transcript yields the observed
15~/o basal-level readthrough (approx. 3% readthrough is observed even when-antiterminator formation is precluded). If 24% of ribosomes release
from the trpLep57A,62A UGA codon prior to comFollowing the convention of Crick & Brenner
(1967), a ( - ) ribosome frameshift is "a shift to the
right", that is, to the 3' side, and a (+) ribosome
frameshift is "a shift to the left."
pletion of 3:4 RNA hairpin synthesis, this model
would predict approximately 12% termination, or
88~/o readthrough. This calculation agrees well with
the 91 ~o readthrough we estimated for trpLep57A,62A (Table 4).
Thus, our data, taken together with that of
Roesser et al. (1989), strongly support an attenuation mechanism in which a ribosome positioned at
the leader peptide stop codon ordinarily blocks antiterminator formation, and occasional release of the
ribosome sets basal level readthrough at the
attenuator. This view differs fl'om an alternative
model in which immediate ribosome release at the
leader peptide stop codon permits rapid re-formation of the 1:2 RNA hairpin to block antiterminatot formation. In such a model the 1:2 hairpin
serves the function of "protector" against antiterminator formation (Keller & Calvo, 1979) and basal
level readthrough would be dictated by occasional
failure of the "protector" to form (Kolter &
Yanofsky, 1984). To the extent that the attenuation
mechanism is similar" in all amino acid biosynthetic
operons, it seems likely that the most important
function of the first RNA hairpin structure in the
leader transcript is to direct RNA polymerase pausing to permit synchronization of transcription and
translation during attenuation.
The consequences, of codon changes in the leader
peptide coding region reported here demonstrate the
intricacies of information content on regulation by
attenuation in bacterial amino acid biosynthetic
operons. Additional manipulation of the length and
composition of the trpL leader peptide coding region
should provide a more complete understanding of
the consequences of ribosome positioning and movement on the events of transcription.
We thank Jim Roesser fbr helpful comments on tile
manuscript and Donna Natalie for" assistance with preparation of polymerase-chain-reaction-amplified DNAs
and instruction in the use of the AMBIS~ radioanalytic
imaging system. This work was supported by grants from
the United States Public Health Service (GM-38660 to
R.L. and GM-09738 to C.Y.) and an award from the
Searle Scholars Program to R.L.C.Y. is a Career
Investigator of the American Heart Association.
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Edited by M. E. Gottesman