Expanding the Genetic Code

15 Pages·2001·0.53 MB·English

doi:10.1006/jmbi.2001.4518 available onlineat http://www.idealibrary.com on J. Mol.Biol. (2001) 307,755–769
Expanding the Genetic Code: Selection of Efficient
Suppressors of Four-base Codons and Identification
of ‘‘Shifty’’ Four-base Codons with a Library
Approach in Escherichia coli
Thomas J. Magliery2, J. Christopher Anderson1 and Peter G. Schultz1*
1Department of Chemistry, The Naturally occurring tRNA mutants are known that suppress (cid:135)1 frame-
Scripps Research Institute shift mutations by means of an extended anticodon loop, and a few have
10550 N. Torrey Pines Rd., La been used in protein mutagenesis. In an effort to expand the number of
Jolla, CA 92037, USA possible ways to uniquely and efficiently encode unnatural amino acids,
we have devised a general strategy to select tRNAs with the ability to
2Department of Chemistry
suppress four-base codons from a library of tRNAs with randomized 8
University of California
or 9 nt anticodon loops. Our selectants included both known and novel
Berkeley, CA 94720, USA
suppressible four-base codons and resulted in a set of very efficient, non-
cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU
and CUAG. The most efficient four-base codon suppressors had Watson-
Crick complementary anticodons, and the sequences of the anticodon
loops outside of the anticodons varied with the anticodon. Additionally,
four-base codon reporter libraries were used to identify ‘‘shifty’’ sites at
which (cid:135)1 frameshifting is most favorable in the absence of suppressor
tRNAs in Escherichia coli. We intend to use these tRNAs to explore the
limits of unnatural polypeptide biosynthesis, both in vitro and eventually
in vivo. In addition, this selection strategy is being extended to identify
novel five- and six-base codon suppressors.
#2001AcademicPress
*Corresponding author Keywords: four-base codon; suppressors; genetic code; shifty codons
Introduction stimulated by such elements as upstream rRNA-
binding motifs, stem-loop structures and under-
used codons. Indeed, these programmed shifts can
Pioneering work by Crick, Brenner and co-
be critical to protein production, e.g. the gag-pol
workers in the early 1960s established the triplet
polyprotein of Rous Sarcoma virus, the Ty1 and
nature of the genetic code (Crick et al., 1961), and
Ty3 elements of yeast, the dnaX gene of DNA Pol
40 years later only rare exceptions are known to
III tau and gamma units, and the self-regulating
the universal correspondence of mRNA codon to
RF2 protein in Escherichia coli (Gesteland et al.,
amino acid (Fox, 1987). However, it has also
1992).
become clear that frame maintenance during trans-
lation is not absolute (Atkins et al., 1991; Kurland, As early as 1968 it was recognized that frame-
1992; Parker, 1989). In addition to the inherent shift mutations could be externally suppressed
accuracy limits of the ribosome in maintaining (Riyasaty & Atkins, 1968). Shortly thereafter,
frame, shifts can be promoted by mutant tRNAs Yourno & Tanemura (1970) showed that protein
and can occur with high frequency at ‘‘pro- from a suppressed frameshift was of wild-type
grammed’’ sites in mRNA. Frameshifts can be sequence, and Riddle & Carbon (1973) showedthat
theexternalframeshiftsuppressor sufD inSalmonel-
la is tRNAGly with a four-base anticodon CCCC
Present address:T. J.Magliery,Department of instead of the wild-type CCC. (Note that we adopt
Chemistry, The Scripps Research Institute, 10550N.
the convention herein of writing both codon and
Torrey PinesRoad, LaJolla, CA 92037, USA.
anticodon sequences in the 50 to 30 direction.) These
T.J.M.and J.C.A.contributed equally tothis work.
experimentsconclusively demonstrated that certain
Abbreviations used: .
E-mailaddress ofthe corresponding author: tRNAs with extended anticodons can read non-tri-
[email protected] plet codons. Most of the known four-base codon
0022-2836/01/030755–15$35.00/0 #2001AcademicPress
756 Selection ofEfficientSuppressors ofFour-base Codons
suppressors have subsequently been isolated from merely render the tRNA less capable of three-base
Salmonella or yeast and are largely single-base decoding and in the process make room for other
insertion mutants of tRNAPro, tRNAGly or tRNALys tRNAs to promiscuously interact with the codon
reading CCCX, GGGX or AAAX, where it is not by a slippage mechanism. For example in Salmonel-
always known which X can be read. The known la, mutant tRNAPro with a GGGG anticodon is
four-base codon suppressors tend to act with effi- methylated in such a way (underlined G37) that
ciencies from very poor to approaching 20% pairing with the mRNA cytosine is obstructed.
(Atkins et al.,1991). Apparently, the tRNAPro with cmo5UGG antico-
Less well known are (cid:135)1 suppressors with more don, which typically reads CCA, CCG and CCU,
extended anticodon loops. For example, the Salmo- can read CCC in the absence of its normal cognate
nellasufT621 derivativeoftRNAArgcausesinsertion tRNA , but favors slippage since there are only
GGG
of arginine in response to CCGU. A synthetic two canonical base-pairs.
mutant of this tRNA, which bears a spontaneous Four-base codon suppression has begun to be
mutation in the DHU loop of unknown import- used for in vitro protein engineering experiments,
ance, has a nine-base anticodon loop that causes initially by Ma et al., to insert alanine at UAGG
(cid:135)1 frameshifting (Tuohy et al., 1992). The only and AGGU with complementary anticodons
other known tRNAs with nine-base anticodon engineered into tRNAAla (Kramer et al., 1998; Ma
loops mediating (cid:135)1 frameshifting are the related et al., 1993). Sisido’s group has used extended-
tRNAPro-derived yeast suppressors SUF7, SUF8, anticodon tRNAs to insert unnatural amino acids
SUF9 and SUF11, which lack a 31:39 anticodon into proteins in vitro, employing known suppres-
stem pair (Atkins etal., 1991). sible four-base codons such as AGGU and
The specificity of pairing is not entirely predict- CGGG (Hohsaka et al., 1996; Hohsaka et al.,
able for extended-anticodon tRNAs. For example, 1999b; Murakami et al., 1998). Significantly,
when the suppression of GGGN codons by tRNAs Hohsaka et al. (1999a), used both of these
bearing NCCC anticodons (SUF16 derivatives) was codons together in a single transcript to insert
analyzed,itwas found thatthe only non-functional two different unnatural amino acid residues into
N:N pair was G:G. This result indicates that the same protein site-specifically.
Watson-Crick pairing at the fourth codon nucleo- In an effort to develop improved tRNA/four-
tide is even less strictly enforced than at the third base codon pairs for protein mutagenesis, Moore
‘‘wobble’’ position for normal codons. However, et al. examined the ability of Su6 (amber tRNALeu)
the identity of the N:N pair does affect suppression derivatives with NCUA anticodons to suppress
efficiency, and canonical pairings in the fourth pos- UAGN codons and also monitored decoding in the
ition gave the strongest suppression (Gaber & (cid:255)1 and 0 frames. In an RF1-deficient E. coli strain,
Culbertson, 1984). Additionally, it has been found UCUA suppressed the UAGA codon with up to
that sufJ, a modified tRNAThr with anticodon 26% efficiency with little decoding in the 0 or (cid:255)1
UGGU, is capable of decoding ACCC and ACCU frames(Mooreetal.,2000). Thispairwasalsofairly
in addition to ACCA (ACCG was not tested; Bossi efficient in the Curran & Yarus study, and differ-
& Roth, 1981). ences between the results of independent exper-
In an engineered system, Curran & Yarus (1987) iments are likely due, in part, to the extent of
tested the ability of derivatives of the glutamine- aminoacylation and maturation with different
inserting amber suppressor Su7 with NCUA anti- tRNA scaffolds. It is clear that decoding four-base
codons to suppress UAGN codons. While the codons with complementary anticodons is
canonical pairs tended to suppress well, some generally favorable, but the rules that govern the
non-canonical pairs were also efficient. For comparative suppression efficiencies for different
example, the best pair arose from a CCUA antico- four-base codons remain unclear.
don and a UAGA codon; also, the GCUA tRNA Likewise,itisnotexhaustivelyknown whatgov-
preferred UAGA and UAGG to its canonical erns the likelihood of frameshift in the absence of
UAGC.Theabilitytoreadafour-basecodonversus suppressor tRNAs. Atkins and co-workers estab-
a three-base codon for these extended-anticodon lished the generality of ‘‘leakiness’’ in frame main-
tRNAs was also assessed, by using tRNAs that tenance by measuring the weak activity from
decode four-base codons whose first three bases insertion and deletion mutants of the gene for
encode a stop codon. In this case there is no com- b-galactosidase induced by the acridine derivative
petitive translation with a canonical tRNA. In gen- ICR-191D. Sixteen mutants varied in activity by
eral, four-base decoding predominated when a 100-fold, with the strongest 0.06% of wild-type
fourth Watson-Crick pair was possible and was (Atkins et al., 1972). This, and studies of immuno-
less likely to predominate otherwise. The authors precipitated truncation products of b-galactosidase
proposed that this is a consequence of mRNA- (Manley, 1978) place the average frequency of fra-
induced stabilization of a tRNA conformation that meshift in the range of 5(cid:2)10(cid:255)4 per translocation
favors four-base decoding, possible only when a event. However, factors such as ribosomal
fourth canonical base-pair is present (Curran & mutation (rpsL and ram), streptomycin treatment,
Yarus,1987). amino acid starvation and ‘‘hungry’’ codons
In contrast, Qian et al. (1998) have proposed that, greatly increase the frequency of frame errors
at least in some cases, extended-anticodon tRNAs (Atkins et al., 1972; Weiss & Gallant, 1983; Weiss
Selection of EfficientSuppressors ofFour-base Codons 757
et al., 1988a). For example, in E. coli, tandem repeat to high concentrations of ampicillin (100-
of AGG, a rare arginine codon read by a minor 2000 mg ml(cid:255)1). Invariably, sequencing of these
tRNA, results in (cid:135)1 frameshift up to 50% of the clones showed that the b-lactamase genes con-
time(Spanjaard& van Duin, 1988). tainedonlythree nucleotides at the sites of interest,
The best-known ((cid:135)1 and (cid:255)1) frameshifting corresponding to Ser and Cys at the conserved S70
events, both programmed and non-programmed, site and corresponding to all amino acids except
occur because of single-base repeats constituting Cys, Met, Pro, Trp and Tyr at the permissive S124
a ‘‘slippery run’’ of mRNA. For example, the (cid:135)1 site (although this is probably largely due to
frameshift that avoids the UGA stop codon at the undersampling of three-base sequence space, since
programmed site in RF2 occurs at the sequence the amount of contamination was small). Presum-
CUUU, mediated by the CUU-decoding tRNALeu ably, these ‘‘deletion’’ mutants, which occur at
(Weiss et al., 1987). While it is easy to understand rates much higher than the 10(cid:255)6 upper-limit esti-
how a long repeat is a frame-maintenance conun- mated for deletion in vivo (Cupples et al., 1990), are
drum for the ribosome, it is equally striking that the result of inefficiency in the coupling and cap-
high levels of shifting are observed only in the pre- ping reactions employed during synthesis of the
sence ofother‘‘stimulatory’’elements(Atkinsetal., oligonucleotides used to generatethe libraries.
1990), such as a Shine-Delgarno-like sequence To remove this contamination, cells transformed
upstream and a UGA stop codon downstream of with the reporter libraries were grown in small
the RF2 shift site (Weiss et al., 1987, 1988b); an batches and analyzed on agar plates for moderate
RNA-pseudoknot downstream of the shift site for levels of ampicillin resistance (100 mg ml(cid:255)1). By
the F2 protein of coronavirus IBV (Brierley et al., pooling the cultures that contained no ampicillin
1989); and the rare AGG codon adjacent down- resistance at this level, uncontaminated, over-
stream to the CUU slip site in yeast Ty elements sampled reporter libraries were obtained (about
(Belcourt &Farabaugh,1990). 5000 clones for the S124(N4) library and 10,000
We were interested in employing a library clones for the S70(N4) library). Sequencing of 48-96
approach to examine all possible four-base codons clones from each reporter library showed that the
for their propensity to shift and, moreover, to libraries were random at each nucleotide and suffi-
exhaustively identify tRNAs that will efficiently ciently unbiased that unique four-base codons
suppress four-base codons. To this end, we have were pulled out only once or twice among these
installed the sequence NNNN into various codons clones.
of the gene for b-lactamase and selected with Because the b-lactamase reporter libraries were
ampicillin in the presence and absence of deriva- constructed from pBR322-dervived vectorscontain-
tives of tRNASer with randomized, extended (eight ing ColE1 origins, tRNA suppressor libraries were
2
or nine nucleotide) anticodon loops. We have suc- constructed with pACYC184-derived vectors with
cessfully identified at least four non-interacting p15a origins to permit co-maintenance within a
sets of efficient four-base codon-tRNA pairs, and single bacterium. These libraries consisted of
we have defined a strikingly simple pattern that derivatives of tRNASer with the anticodon loop (7
2
underlies ‘‘shiftiness’’ in four-base codons. These nt)replacedwitheightorninerandom nucleotides,
pairs will be used in our ongoing efforts to engin- transcribed under the control of the strong lpp pro-
eer bacteria capable of inserting unnatural amino moter and efficient rrnC terminator. This tRNA
acid residues into proteins. was chosen because seryl-tRNA synthetase (SerRS)
does not recognize the anticodon loop of tRNASer.
Major recognition elements are, instead, in the long
Results
variable arm, acceptor stem, and the D and T(cid:9)C
loops, where the SerRS contacts the tRNA (Biou
Library construction and characterization
et al., 1994; Price et al., 1993). The choice of a tRNA
Since the basis of four-base codon suppression is scaffold and cognate synthetase that is permissive
poorly understood, we adopted a combinatorial to anticodon loop structure ensures that suppres-
approach to examine the suppression of all poss- sionefficiencyisrelatedtocodon-dependenteffects
ible four-base codons by all possible appropriately rather than the effects of variable aminoacylation
sized tRNA anticodon loops. Two types of libraries with different anticodon sequences. For each of
were generated: four-base codon reporters and these libraries, 24 clones were sequenced and
tRNA suppressors (Figure 1). The reporter system found to beuniqueand unbiased.
involved replacement of a conserved (S70) or per-
missive (S124) codon in the gene for b-lactamase
Selected four-base codons at S124 and S70
with four random nucleotides. These libraries,
which each contain 44(cid:136)256 unique members, bear The libraries were crossed by the transformation
an insertion mutation at the site of interest, produ- of the appropriate tRNA library into competent
cing an inactive enzyme as a result of the frame cells of the reporter strains, followed by selection
alteration that renders transformed bacteria incap- on media containing various concentrations of
able of survival on ampicillin. To our surprise, ampicillin. Survival on higher concentrations of
however, approximately 0.5% of the members of ampicillin requires that more b-lactamase be pre-
these reporter libraries survived on moderate sent in the cell, which is related to the extent to
758 Selection ofEfficientSuppressors ofFour-base Codons
Figure 1. Schematic of b-lacta-
mase libraries and tRNASer
libraries. Insertion of NNNN at
Ser70 or Ser124 results in abortive
translation unless the frameshift is
suppressed.
which the tRNA in the selectant is able to mediate lytic Ser of TEM-1 b-lactamase and requires Ser or
frameshift at the site of mutation in the reporter. Cys for activity, confirms that these four-base
Thus, survival at the highest levels of ampicillin codons are suppressed by our modified seryl-
indicates the presence of tRNAs that read the co- tRNA.
transformedfour-basecodons most efficiently. Since there was no apparent pattern to the
The library of 8 nt anticodon loop tRNAs was sequences of the suppressed four-base codons, we
first crossed with the S124 and S70 reporter devised an experiment to examine whether or not
libraries, choosing two sites to examine the effects any four-base codon could be suppressed, at least
of mRNA context. In addition to different general at low levels. Six clones from the S70(N4) reporter
contexts in the gene, these sites also have different library were selected at random and crossed
local contexts with different nucleotides 50 and 30 against the tRNASer(N8) library. From these clones
to the four-base codon (Figure 1). At the permiss- (Table 2), tRNAs that efficiently suppress UUCU,
ive S124 site, four-base codons selected at moder- GGAU and CGGA to at least 200 mg ml(cid:255)1 ampicil-
ate ampicillin concentrations (200-300 mg ml(cid:255)1) lin could be found. AAUG and ACGC suppressors
included a subset of AGGN, CCCN, CGGN, were generally much weaker, between 5 and 10 mg
GGGN and UAGN, for which four-base codon ml(cid:255)1 ampicillin. A suppressor for UGAA, based on
suppressors are known, as well as CUAG, UCAA, the UGA stop codon, could not befound, however,
CCUU, CUCU, GGAC and UGCG (Table 1). Only even at 5 mgml(cid:255)1ampicillin.
one of these, AGGA, could be suppressed at The procedure for removal of the three-base con-
1000 mg ml(cid:255)1 ampicillin. At the S70 site, however, tamination at the catalytic S70 site cannot remove
AGGG, CUCU and UAGA were also suppressed those clones that contain a random three-base
at 1000 mg ml(cid:255)1, and more codons were rep- codon that corresponds to a missense mutation (i.e.
resented at the 300 mg ml(cid:255)1 level than for the S124 anything other than Ser or Cys). As a result, upon
site. Suppression of the S70 site, which is the cata- crossing this library with the tRNA library, a num-
Selection of EfficientSuppressors ofFour-base Codons 759
Table 1. Selectedfour-base codons at S124and S70at high and moderateampicillin concentrations
S124 S70
1000mgml(cid:255)1 300mgml(cid:255)1 1000mgml(cid:255)1 300mgml(cid:255)1
AGGA AGGA AGGA AGGA
AGGG AGGG AGGC
AGGC CUCU AGGG
CCCU UAGA AGGU
UAGA CCAU
CGGG CCCC
UCAA CCCU
CGGC
CUAC
CUAG
CUCU
UAGA
ber of clones were isolated that contained missense suppressors contain 8-nt anticodon loops or 7 nt
three-base codons that were presumably being (i.e. normal) loops as a result of the same type of
suppressed by the Ser-inserting tRNA library ‘‘deletion’’ seenin thereporter libraries.
member. Interestingly, the most efficiently sup-
pressed, AGG and CGG at 1000 mg ml(cid:255)1, corre-
spond to the first three nucleotides of some of the Sequence variation of tRNA suppressors of
most efficiently suppressed four-base codons four-base codons
(Table 3). It was not determined whether the tRNA
In order to examine the diversity of tRNA antic-
odon loops that could suppress each selected
codon, the reporter plasmid was isolated and the
tRNASer(N8) library was re-crossed against indi-
Table 2. Selection of tRNA suppressors of randomly vidual reporter sequences at S124. These were then
chosen codons selected at a variety of levels of ampicillin for each
reporter, and the tRNA sequences were examined.
Maximumsuppression
Codon level tRNAsequences In each case, suppression at the highest levels was
mediated by a tRNA bearing the complementary
UGAA >5 Nonedetected
anticodon (Table 4). Interestingly, the sequences
AAUG 5-10 CUCGUUAC
CUCGUUAA external to the anticodon (i.e. the two nucleotides
ACGC 5-10 CUGCGUAU on either side of the anticodon) converged on
CUACGUAU
GGACGUAA
UUCU >200 UUAGAAAG
AUAGAAAG
ACGGAAAC Table 3. Codons for which Ser missense suppressors
UUAGAAAC were slected at S70
CUAGAAAC
ACAGAAAC [Amp] Codon Acid
CGGGAAAA
GGAU >200 GUGUCCAU 1000 AGG Arg
CAGUCCAU CGC Arg
UGAUCCAU 300 ACA Thr
CCAUCCAG AGG Arg
UAGUCCAC AUA Ile
UCAUCCAC CCA Pro
CCAUCCAC CCU Pro
UAAUCCAC CGA Arg
AAGUCCAA CGG Arg
CGGA >200 CGGCCCGG UUG Leu
UUUCCGUA 100 ACG Thr
CCUCCGUA AUA Ile
GUUCCGGC CCU Pro
CUUCCGCU CGA Arg
ACUCCGCG CGG Arg
GUUCCGGC CUC Leu
ACUCCGUA GCC Ala
AAUCCGCU GGC Gly
ACUCCGUA GUG Val
ACUCCGUU UUA Tyr
Suppressionlevelsareinampicillinconcentration,mgml(cid:255)1. Ampicillinconcentrationsareinmgml(cid:255)1.
760 Selection ofEfficientSuppressors ofFour-base Codons
Table 4. Selectants from re-cross of individual four-base Watson-Crick complementarity to the first
codons againsttRNASer(N8) three bases of the codon is most critical, followed
by complementarity at the fourth base, the pre-
sence of a purine at position 37 (30 to the antico-
don) and the presence of a U at position 33 (50 to
the anticodon).
Analogously, to examine how efficiently AGGA
could be suppressed by 9-nt anticodon loop
tRNAs, the AGGA reporter at S124 was crossed
against tRNASer(N9) and selected at 20 and 300 mg
ml(cid:255)1 ampicillin. At 300 mg ml(cid:255)1ampicillin, the con-
sensus sequence was BNKUCCUAH, where K(cid:136)G
or U and H(cid:136)A or C or U (Table 6). However, at
20 mg ml(cid:255)1, the consensus sequence degenerated to
NNNUCCUAN. In a single case out of 38 clones,
CUUCCUAUU, the extra nucleotide in the loop
was on the 30 side of the anticodon (UCCU). At
both levels of ampicillin selection, tRNAs with 8 nt
anticodon loops were among the selectants, pre-
Ampicillin survival levels are in mg ml(cid:255)1 for codons at S124.
sumably as a result of ‘‘deletion’’ from inefficient
ND(cid:136)notdetermined.
aEven at the highest levels of ampicillin selection, three capping during oligonucleotide synthesis seen in
tRNAs were found for the AGGA anticodon: A...A, C...A, the reporter libraries. These deletions dominate the
C...C. selection at higher ampicillin levels, indicating that
bDeterminedatS70.
suppression of four-base codons (or at least
AGGA) is more robust with tRNAs bearing 8 nt
anticodon loopsrather than 9-nt loops, overall.
different sequences depending upon the anticodon.
Specificity of tRNA suppressors of four-
To examine the effect of sequence variation in the
base codons
anticodon loop outside of the anticodon per se,
tRNAs from the tRNASer(N8) library were selected The most robust suppressors of each of the most
at different levels of ampicillin (20 mg ml(cid:255)1 to favorable codons were isolated and re-crossed
1500 mg ml(cid:255)1) against a single reporter codon at against the S70(N4) reporter library with selection
S124 (Table 5). For this experiment, we selected the at very low ampicillin (10 mg ml(cid:255)1). Here, S70 was
most efficiently suppressed codon (AGGA) to selected, since the site is slightly easier to suppress,
examine the widest array of suppression efficiency. and we were interested in examining extremely
At 1500 mg ml(cid:255)1 ampicillin, the sequences con- low levels ofsuppression, ifthey existed. Selectants
verged on MUUCCUAM, where M(cid:136)A or C. At were then transferred to increasingly higher con-
300 mg ml(cid:255)1, the convergence was reduced to centrations of ampicillin and sequenced to identify
NBUCCURN, where B(cid:136)C or G or U and R(cid:136)A the extent to which each tRNA is capable of
or G. At 20 mg/ml, the consensus was reduced to suppressing other four-base codons. In each case,
NNNCCUDN, where D(cid:136)A or G or U, with loss suppression of the canonical four-base codon was
of canonical recognition of the fourth base of the highly favored, with suppression of related codons
codon but maintenance of the other three bases. with non-canonical pairing at the fourth codon
This allows us to rank the different anticodon pos- position possible at fivefold lower ampicillin at
itions in importance for suppression efficiency: best (Table 7). The tRNA that efficiently reads
Table 5. Selectedeight-nt anticodon looptRNAs to suppress AGGA atmoderate and low ampicillin levels
300mgml(cid:255)1 20mgml(cid:255)1
ACUCCUAA CUUCCUAC AAUCCUAG CAUCCUAU UAUCCUGU
ACUCCUAC GGUCCUAU AAUCCUAU CGGCCUAU UGUCCUAG
AGUCCUAC GUUCCUAA ACUCCUAU CGUCCUGU UGUCCUAU
AGUCCUGC UCUCCUAA ACGCCUAU CGUCCUUU UGUCCUGC
AUUCCUAG UCUCCUAC ACUCCUAU CUACCUAU UUUCCUAU
CCUCCUAA UUUCCUAC AGGCCUGG CUUCCUAA UUACCUAA
CCUCCUAC UUUCCUAG AGUCCUAC GGUCCUGA UUUCCUAG
CGUCCUAU UUUCCUAU AGUCCUUG GUUCCUAA UUUCCUAU
CUUCCUAA AUUCCUAG GUUCCUAU UUUCCUGU
CAACCUAA GUUCCUGU
CAUCCUAA UAUCCUAA
Sequenceswerefoundonetosixtimeseach.Onlytheanticodonloopsequenceisshown.
Selection of EfficientSuppressors ofFour-base Codons 761
Table 6. Selectednine-nt anticodon looptRNAs to suppress AGGA at moderateand low ampicillin levels
300mgml(cid:255)1 20mgml(cid:255)1
CAUUCCUAU AAUUCCUAC CCUUCCUAA GAUUCCUAU
CCUUCCUAU ACGUCCUAC CCUUCCUAU GCUUCCUAU
CUGUCCUAA ACUUCCUAC CUGUCCUAC UCGUCCUAG
GCUUCCUAU ACUUCCUAU CUGUCCUAU UCUUCCUAA
GGUUCCUAU AGUUCCUAC CUUUCCUAA UCUUCCUAG
UUGUCCUAC AUUUCCUAA CUUUCCUAU UUGUCCUAC
AUUUCCUAC GACUCCUAC UUUUCCUAC
CACUCCUAC GAGUCCUAC UUUUCCUAU
CCAUCCUAU GAUUCCUAC CUUCCUAUU
Sequenceswerefoundonetofourtimeseach.Onlytheanticodonloopsequenceisshown.
AGGA (1500 mg ml(cid:255)1 ampicillin), for example, ing at of the four-base codons at S124 was no more
reads AGGU to only 100 mg ml(cid:255)1, and AGGG and than twice the background hydrolysis by TOP10
AGGC to only 50 mg ml(cid:255)1. In contrast, suppression E. coli (0.8-1.7 units versus 0.9 unit), which was
of no other codon could be detected for the tRNA comparable to readthrough of the amber stop
that suppresses CUAG. codon (1.2 units, Figure 2). Suppression of the
four-base codons was in the range of 17 to 240
units, or 2.5-35% of wild-type (AGT or b-lactamase
Efficiency of frameshift mediated by four-base on pBR322). However, in this assay, the very-effi-
codon suppressors cient supD suppressor of the amber stop codon
exhibited virtually the same b-lactamase activity as
The level of readthrough of the four-base codons
observed in cells with AGT (Ser) at the S124 site.
and the efficiency of suppression of these codons
This may indicate that it is possible to saturate the
were quantified by determining the amount of
amount of b-lactamase that is exported to the peri-
b-lactamase per cell with the chromogenic sub-
plasm in this system. Therefore, while the activities
strate nitrocefin. Convenient arbitrary units were
allow us to compare frameshift efficiencies, we
defined to normalize the activity for the number of
cannot strictly correlate these activity data with
cells assayed and to subtract out the effects of the
suppression efficiencies as compared to translation
slow background hydrolysis of nitrocefin (see
of wild-type b-lactamase.
Materials and Methods). Spontaneous frameshift-
Identification of ‘‘shifty’’ codons
It was found empirically during the process of
Table 7. Codons selected by most efficient four-base
removing three-base contamination from the
codon suppressors
libraries that no four-base codon was read through
bythenaturaltranslationalmachinery atlevelssuf-
ficient to permit survival at 100 mg ml(cid:255)1 ampicillin.
However, at lower levels of ampicillin, some four-
base codons can be read at low levels, or are inher-
ently shifty (Table 8). Strikingly, each of these four-
Table 8. ‘‘Shifty’’ four-base codons detected at the S124
site
25mgml(cid:255)1 10mgml(cid:255)1
AAAA AAAA
AGGC AGGC
CGGC CGAU
CUUA CGGA
CUUU CGGC
GGGA CGGG
UUUC CGGU
UUUU CUUA
CUUC
CUUU
GGGA
Sequences for the anticodon loop are shown. Codon in par- UUUC
entheses is that which elicited the tRNA in selection against a UUUU
single reporter (Table 3). Suppression levels are indicated for
eachtRNA/four-basecodonpairinmgml(cid:255)1ampicillin. Selectionlevelsaregiveninampicillinconcentration.
762 Selection ofEfficientSuppressors ofFour-base Codons
Figure 2. Suppression efficiency of four-base codon/tRNA pairs measured by nitrocefin turnover. See Materials
and Methods for definition of units. Readthrough of UAG, AGGA, CCCU, CUAG and UAGA was 0.8-1.7 units,
while TOP10 cells alone exhibit about 0.9 unit. Suppression of four-base codons resulted in 17-240 units of activity,
compared to 970 for suppression of UAG by supD. Results are the average of three to five independent trials per
data point.
base codons has a tandem repeat in the central two codon in the E. coli genome (Inokuchi & Yamao,
nucleotides (a purine repeat in one case). It is also 1995). Nevertheless, the best suppressors do not
noteworthy that some of the best suppressed simply correspond to the least represented tRNAs
codons (e.g. AGGC, CGGN and CGAU) but not all or codons, since we found no robust suppressors
of them (e.g. AGGA) are among this shifty set. For of highly underrepresented codons AGAN, AUAN
some codons (e.g. AGGN, AAAN, and GGGN), or CGAN. Also, among randomly selected codons
only a subset of the family is present, indicating that vary in usage from overrepresented (AAUG
that slipping at a particular three-base codon is and UUCU) to moderate representation (ACGC) to
strongly influenced by the base that follows. Some underrepresented (GGAU and CGGA), and that
of these, such as AGGA, are probably only slightly lack any evident sequence pattern, weak to moder-
less shifty (5 mg ml(cid:255)1), but it becomes difficult to ate suppressors could be found (Table 9). Of
examine libraries at this low level of selection due course, the procedure employed here selects for
to backgroundsurvival of satellites on ampicillin. growth on ampicillin and therefore requires both
an efficient four-base codon/tRNA interaction and
a lack of toxicity to the cell. We cannot rule out the
Discussion
possibility of efficient but toxic suppressors of
highlyrepresented four-basecodons.
Suppression of four-base codons
The identity of the fourth base has a profound
When the first frameshift suppressors were iso- effect on the ability of the four-base codon to be
lated, it was thought that only repeating codons suppressed. For the AGGN family, the series
like GGGG could be suppressed. However, a num- appears to be AGGA>AGGG>AGGC(cid:25)AGGU
ber of exceptions, like ACCN and UAGN, were for canonical suppressors. However, Hohsaka et al.
later found or engineered (Atkins et al., 1991). (1996), saw little difference in the in vitro suppres-
Among those efficient suppressors identified in sion levels among these four codons using a yeast
this study, it is clear that there is no sequence pat- tRNAPhe scaffold, perhaps due to the extreme over-
tern that generally underlies efficient four-base expression conditions he employed in contrast to
suppression. For example, CUAG is completely the moderate, natural b-lactamase promoter
non-repetitive, and GGGG, AAAA and UUUU are employed here. Likewise, only UAGA of UAGN,
not among the best suppressors. However, the and CGGC and CGGG of CGGN, are among the
most efficiently suppressed four-base codons corre- most strongly suppressed codons.
spond to three-base codons (plus one 30 nucleotide) Some of the four-base codons that we identified
withlow usage and low tRNA abundance in E. coli were already known, including AGGN, used in
(Table 9). For example, AGG is the least used protein engineering (Hohsaka et al., 1996; Ma et al.,
Selection of EfficientSuppressors ofFour-base Codons 763
Table 9. Codon usage and tRNA abundance for codons suppressible with 8 nt anticodon loop tRNAs and ‘‘shifty’’
codons
tRNAabundance S124suppressionwith S124readthrough
Codon(acid) Codonusage(0.1%)a (relative)b tRNA (withouttRNA)
AGG(Arg) 0.9 ND (cid:135) (cid:135)
CCA(Pro) 7.4 ND (cid:135)
CCC(Pro) 3.4 ND (cid:135)
CGG(Arg) 3.6 Minor (cid:135) (cid:135)
CUA(Leu) 2.5 Minor (cid:135)
CUC(Leu) 9.3 0.30 (cid:135)
UAG(amber) ND N/A (cid:135)
UCA(Ser) 5.6 0.25 (cid:135)
AAA(Lys) 38.4 1.00 (cid:135)
CGA(Arg) 2.6 0.90 (cid:135)
CUU(Leu) 9.9 0.30 (cid:135)
GGG(Gly) 8.6 0.10 (cid:135)
UUU(Phe) 16.9 0.35 (cid:135)
AAU(Asn) 14.8 0.60 weak
ACG(Thr) 11.4 ND weak
GGA(Gly) 5.5 0.20 weak
UUC(Phe) 19.1 0.35 weak
aUsageinE.coli.Ifallsensecodonswereusedequally,usagewouldbe16.4.
bRelativetotRNALeuwithcodonusage56.3.
3
ND,nodata.N/A,notapplicable.Minor,abundancetoolowforaccuratemeasurement(Inokuchi&Yamao,1995).
1993),CCCU, which isSUF8 (Cummins et al., 1985) there is no special pattern underlying the sup-
and UAGA (Curran & Yarus, 1987; Moore et al., pressed codons, as might be expected if slippage
2000). However, CUAG is apparently a completely were occurring.
novel efficiently suppressed four-base codon, con- Diversity among tRNAs that suppress four-base
sistent with the fact that CTAG is underrepre- codons can also be assessed by comparing the
sented in the bacterial genome (Burge et al., 1992). tRNA sequences selected with a single reporter
These suppressors acted with efficiencies of 2.5- codon. This approach gives us some idea about the
35% of the maximum activity detectable with a tolerance of the E. coli translational apparatus to
nitrocefin-based assay (AGU or supD/UAG), variation in the tRNAs for suppression of four-
which is consistent with activities from the most base codons (presumably combined with tRNA
efficient natural and engineered suppressors maturation and aminoacylation differences, which
known. Also, fairly efficient suppressors for the are probably minimal here). Figure 3 shows the
four-base codons CUAC, CCAU, UCAA, CCUU consensus sequences for tRNAs able to suppress
and CUCU were found (Tables 1 and 4). Examin- AGGA at various levels of ampicillin compared
ation of the anticodon loop sequences shows that with the known E. coli tRNAs. The best suppres-
the best tRNAs for each codon have a canonical sors of this codon maintain the universal U33 and
anticodon, but the rest of the loop does not necess- highly favored A37 seen in E. coli tRNAs. At the
arily converge on the sequence consensus observed 300 mg ml(cid:255)1 ampicillin level, U33 or C33 and A37
in E. coli tRNAs. For example, the tRNA that sup- are favored, but G is present in some clones at
presses CUAG has G33 and G37 instead of U33 both sites. When much weaker suppressors were
and A37, and the tRNA that suppresses CCCU has selected (20 mg ml(cid:255)1 ampicillin), no base is con-
a U37. (See Figure 1 for numbering of nucleotides served at position 33 and, though A37 is favored,
in the 8 nt and 9 nt anticodon loops.) Presumably, G37 and U37 are seen. This calls into question the
these mutations adjust the loop conformation to necessity of the U33 to make the sharp turn seen in
allow maximally favorable interaction between the the X-ray crystal structure of tRNAPhe (Holbrook
codon and anticodon, which is quite possibly et al., 1978; Sussman & Kim, 1976). Even the 33.5
different from the most favorable conformation for position corresponding to AGGA is not entirely
a three-base anticodon. These data support a conserved in these weak suppressors, though
model in which the extended-codon tRNA directly U33.5 is still strongly favored. Some 5% of the
reads all four bases of the codon (as opposed to a clones selected at 300 mg ml(cid:255)1 and 19% at 20 mg
slippage model), since (1) the most efficient sup- ml(cid:255)1 have the possibility of an A:U or U:A pair at
pressors invariably had Watson-Crick complemen- the 32:38 site, which would formally extend the
tarity at all four bases of the codon, (2) the anticodon stem by one pair and leave a 6 nt loop.
suppression at S70 required insertion of a serine or Other work has suggested that it is unlikely that a
cysteine residue, demonstrating that the modified 6 nt loop can present a four-base anticodon (Atkins
tRNASer was delivering the amino acid, and (3) et al., 1991), and five of the seven clones of this
764 Selection ofEfficientSuppressors ofFour-base Codons
Figure 3. Nucleotide representation at anticodon loop sites in E. coli tRNAs and moderate and weak tRNA sup-
pressors of AGGA with 8 nt and 9 nt anticodon loops. Suppression is at S124 and listed in ampicillin concentration
of mgml(cid:255)1.
type retain complementary UCCU anticodons, per- 32:38 pairing with the favored U38. At 300 mg
haps suggesting why only the weaker A:U pair is ml(cid:255)1 ampicillin, three of the nine clones have
seen at this site and not the C:G pair. Nevertheless, the possibility of a weak G32:U38 wobble pair
the possibility of 6 nt anticodon loop tRNAs (resulting in a 7 nt anticodon loop and extended
decoding four-base codons cannot beruled out. anticodon stem), but none can canonically pair.
When this selection scheme was applied to 9 However, among the weaker suppressors, two of
nt anticodon loop tRNAs that suppress AGGA 37 clones can have a U:A or A:U pair and five
(Figure 3), a UCCU anticodon was found in can have a G:C pair. Particularly in the latter
each tRNA and, in every clone but one, the case, it is possible that the 7 nt anticodon loop
extra nucleotide is added 50 to the anticodon (as with an extended anticodon stem is the active
was always the case with the 8 nt anticodon conformation suppressing the four-base codon.
loop tRNAs). At 300 mg ml(cid:255)1 ampicillin, U38 is Indeed, recent work by Auffinger & Westhof
favored, while either pyrimidine is favored at (1999) suggests structural conservation at the
20 mg ml(cid:255)1. This differs both from the E. coli 32:38 position in all tRNAs and excludes G:C
preference for A38 and lack of preference in 8 pairs, perhaps lending credence to the view that
nt tRNAs. U33 is strongly preferred at both this pair is part of the stem, not the loop, in
ampicillin concentrations, much more so than in these cases.
the 8 nt anticodon loops. Also, while there is In addition to exhibiting high suppression effi-
degeneracy at the 32 position, the stronger sup- ciency, any useful four-base codon suppressor
pressors do not have A32, possibly to avoid must also not cross-react with other codons (three