The Rate of Peptidyl-tRNA Dissociation from the Ribosome during Minigene Expression Depends on the Nature of the Last Decoding Interaction*
L. Rogelio Cruz-Vera
,
Elena Hernández-Ramón,
Bernardo Pérez-Zamorano and
Gabriel Guarneros
From the
Departamento de Genética y Biología Molecular, Centro de
Investigación y de Estudios Avanzados del IPN, Apartado Postal
14740, México Distrito Federal 07000
Received for publication, February 3, 2003
, and in revised form, April 16, 2003.
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ABSTRACT
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The expression of some very short open reading frames (ORFs) in
Escherichia coli results in peptidyl-tRNA accumulation that is lethal
to cells defective in peptidyl-tRNA hydrolase activity. In an attempt to
understand the factors that affect this phenotype, we have surveyed the
toxicity of a complete set of two-codon ORFs cloned as minigenes in inducible
expression vectors. The minigenes were tested in hydrolase-defective hosts and
classified according to their degree of toxicity. In general, minigenes
harboring codons belonging to the same box in the standard table of the
genetic code mediated similar degrees of toxicity. Moreover, the levels of
peptidyl-tRNA accumulation for synonymous minigenes decoded by the same tRNA
were comparable. However, two exceptions were observed: (i) expression of
minigenes harboring the Arg codons CGA, CGU, and CGC, resulted in the
accumulation of different levels of the unique peptidyl-tRNAArg-2
and (ii) the toxicity of minigenes containing CUG and UCU codons, each
recognized by two different tRNAs, depended on peptidyl-tRNA accumulation of
only one of them. Non-toxic, or partly toxic, minigenes prompted higher
accumulation levels of peptidyl-tRNA upon deprivation of active RF1, implying
that translation termination occurred efficiently. Our data indicate that the
nature of the last decoding tRNA is crucial in the rate of peptidyl-tRNA
release from the ribosome.
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INTRODUCTION
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Minigenes are DNA sequences present in bacterial chromosomes that may be
expressed into functionally active oligopeptides. In Escherichia coli
for example, translation of a peptide encoded in a minigene present in the 23
S rRNA, turns cells erythromycin resistant
(1); also, peptides containing
five to eight amino acid residues encoded in the attenuator sequence of genes,
which confer resistance to chloramphenicol and erythromycin (catA86,
cmlA and ermC), inhibit peptidyltransferase activity in bacteria
(2).
Translation of two-codon minigenes located in bacteriophage lambda
chromosome bar regions is lethal to cells partly defective in
peptidyl-tRNA hydrolase activity, but not to wild-type bacteria
(3). Translation of
bar minigene mRNAs results in premature release of peptidyl-tRNAs
from ribosomes (a phenomenon called "drop-off"); under limited
Pth1 activity, these
peptidyl-tRNAs accumulate in the cell. It has been proposed that lethality
stems from the subsequent shortage in the pool of specific tRNAs for further
involvement in protein synthesis
(4). Recently, evidence that
seems to support this inference has been obtained for a ribosome bypassing
system (5), but the alternative
explanation that peptidyl-tRNAs might be toxic per se has not been
ruled out (6).
Translation ends at the termination codon in an mRNA, when the ribosomal
peptidyl-transferase presumably hydrolyzes the ester bond between the
completed polypeptide chain and the last tRNA. The termination reaction
requires the concurrence of the release factors RF-1 or RF-2 (depending on the
nature of the termination codon) and other factors catalyzing the release of
the mature protein (7,
8). Drop-off is a normal, if
relatively rare, event in protein synthesis that can occur during elongation
or instead of polypeptide termination
(9,
10). If the rates of
peptidyl-tRNA synthesis and drop-off exceed the rates of termination and Pth
hydrolysis, peptidyl-tRNA accumulates and thus critically reduces the
concentration of aminoacylable tRNA and increases that of peptidyl-tRNAs
(4,
9). The up-shift to
non-permissive temperatures of a thermosensitive pth mutant,
pth(Ts), results in peptidyl-tRNA buildup of all the tRNAs assayed.
The rates of peptidyl-tRNA accumulation differ as a function of the tRNA
species. Thus, families of tRNA cognate to codons for Lys, Thr, and Asn
accumulate the fastest, whereas those cognate to codons for Leu, Gly, and Cys
accumulate the slowest (11).
These results suggest that the drop-off rates depend on the codons
involved.
Toxicity and peptidyl-tRNA accumulation in the pth(Ts) mutant is
alleviated in strains defective for the translation termination factors RF-3
and RRF (12). Drop-off during
minigene mRNA translation is enhanced by these termination factors as well as
the elongation factor EF-G (9).
In vitro experiments with different synthetic minigenes have shown
that the relative rates of termination and drop-off vary according to the
composition of the last sense codon, the nature and context of the stop codon,
and the length of the mini-ORF and that toxicity is correlated to these
conditions (9,
13). In addition, the strong
effect of the SD sequence affects peptidyl-tRNA accumulation by driving
minigene mRNA through several rounds of translation without dissociation from
the ribosome (9). Despite these
results, the scarce number and heterogeneity of minigenes studied so far does
not allow us to draw conclusions about the role of codon composition in
drop-off. We have studied the effect of the last sense codon on peptidyl-tRNA
accumulation, minigene mRNA concentration, translation termination, and
toxicity to pth-defective cells. Our results indicate that the degree
of toxicity stems from the intrinsic inability of certain codon-tRNA complexes
to mediate efficient translation termination.
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EXPERIMENTAL PROCEDURES
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Bacterial Strains and Toxicity AssayThe following E.
coli K-12 strains were used: C600 (thr1 leu6 thi1 supE44 tonA
lacY1), our collection; pth-defective mutants C600rap (C600
pth[rap] zch::Tn10) and C600pth(Ts) (C600
pth[Ts] zch::Tn10)
(14); C600rap Tcs,
tetracycline-sensitive derivative of C600rap
(15); US486 (W3110
prfA1), a thermo-sensitive mutant defective in release factor RF-1
(R. Buckingham collection; Ref.
16); GG05 (US486
pth[rap] zch::Tn10), US486 with bacteriophage P1
(C600rap); and GG06 (C600 pth[rap] prfA1
zch::Tn10), C600rapTcs with P1 (GG05), this work. The
viability experiments to estimate the degree of minigene toxicity were
performed by streaking a single colony of cells harboring the minigene library
on Luria Broth (LB)-agar plates containing 100 µg/ml ampicillin and 1
mM IPTG and incubating at 32 °C for 24 h.
Minigene Library ConstructionThe complete set of 64
constructs harboring two-codon minigenes was obtained by cloning duplex
synthetic oligos into vector pKQV4, which carries the IPTG-inducible
tac promoter (Fig.
1A; Ref.
17). An attempt was made to
generate the 64 pairs of complementary synthetic oligos, namely
5'-AATTCATGNNNTAAATA-3' and
5'-AGCTTATTTANNNCATG-3' (the EcoRI and
HindIII cohesive ends are underlined and in bold, respectively), by
random synthesis at positions NNN corresponding to the variable minigene codon
(Fig. 1A). Duplexes
were obtained by allowing complementary oligos to hybridize in a mixture
containing 400 pmol of each random oligo in 100 µl of buffer (10
mM Tris-HCl, pH 7.2, 1 mM EDTA) for 5 min at 95 °C
and slowly cooled down to room temperature. Duplexes were then cloned into the
vector after their double restriction digestion with both EcoRI and
HindIII. Ligation of inserts into this vector was performed using 40
fmol of duplex mixture with 15 fmol of the double-restricted vector in 20
µl of buffer (10 mM Tris-HCl, pH 7.2, 1 mM ATP, 10
mM magnesium acetate, 25 mM NaCl, and 10 units of T4 DNA
ligase (Amersham Biosciences)) for 1 h at room temperature. The ligation mix
was used to transform C6OOrap cells
(18), and selected clones were
tested for the absence of the SalI site present in the unsubstituted
vector only. Inserts from these clones were sequenced using Big-Dye sequencing
kits (Applied Biosystems), using the forward oligo
5'-GACATAACGGTTCTGGC-3', corresponding to a sequence close to
Ptac, and the reverse oligo 5'-CTGTTTTATCAGACCGC-3',
corresponding to a sequence upstream to the 5 S gene
(Fig. 1A). Thirty-two
minigenes were obtained after random hybridization, and the rest were obtained
by direct cloning of specific duplexes into the expression vector as described
above.

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FIG. 1. Constructs and degree of toxicity of two-codon minigenes.
A, schematic diagram of a linear version of the used constructs: each
construct contained a single minigene cloned between the EcoRI and
HindIII sites (in italics) in pKQV4; the relative positions
of other genetic markers are also indicated. Minigenes were under the control
of the Ptac/Olac transcriptional promoter and were
followed by the transcriptional terminator Trrnb. Translation was
controlled by a SD sequence (underlined) and initiation (ATG) and
stop (TAA) triplets. B, genetic code indicating the variable codon of
the 64 possible two-codon minigene constructs and, for the analyzed cases, the
corresponding tRNAs and amino acid residues (columns 2, 3, and
1, respectively, in each box). The viability of C600pth(Ts)
and C600rap cells transformed with each minigene was measured after IPTG
induction of minigene expression (see "Experimental Procedures").
Different degrees of toxicity are given in different colors: red,
minigenes toxic to both pth-deficient strains; blue, partly
toxic minigenes that inhibit the growth of pth(rap) cells only;
green, non-toxic minigenes. The figures in each box correspond to
estimated peptidyl-tRNA fractions, expressed as a percentage of the
corresponding tRNA present in either C600pth(Ts) (columns
numbered 4) or C600rap (columns numbered 5) after
30 min of expression. Underlined codons are decoded by tRNAs having
concentrations comparable with that of tRNAArg-4, typically
considered as a scarce tRNA species
(22). Codons in
half-brackets are cognate to a unique tRNA.
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Peptidyl-tRNA LevelsPeptidyl-tRNA levels were estimated by
a modification of the Northern blot technique of Varshney et al.
(19). This method is more
specific than the traditional assay
(20), because it distinguishes
individual tRNAs. Briefly, the transformants for minigene constructs were
cultured on LB (100 µg/ml ampicillin) at 32 °C to an
A600 value of 0.5 and induced with 1 mM IPTG
for different periods of time (see "Results") at 32 °C. To
estimate the fraction of peptidyl-tRNA relative to total tRNA, aminoacyl-tRNAs
were hydrolyzed by the cupric reaction
(21). Total extracted tRNA was
resuspended in 20 µl of water and divided into two aliquots: a control
sample that was mixed with 90 µl of 20 mM sodium acetate, pH
5.0, and a hydrolysis sample with 10 mM CuSO4. Each
reaction was incubated for 30 min at 37 °C and was precipitated with 2
volumes of ethanol after addition of EDTA to a final concentration of 5
mM. The samples were resolved by acid/urea PAGE and transferred to
nylon membranes; tRNA-containing species were revealed using specific
32P-labeled oligo probes (5 x 106 cpm/pmol; Refs.
19 and
22). The peptidyl-tRNA
fraction was estimated from the radioactivity ratio between the measured
radioactivities in the (peptidyl-tRNA/peptidyl-tRNA + tRNA) respective bands
as determined in a Typhoon Scan (Amersham Biosciences).
Minigene mRNA DetectionTotal RNA was obtained from cultures
of transformants for minigene constructs induced with IPTG. Minigene mRNA was
revealed by Northern blot analysis using a 32P-labeled DNA probe (2
x 106 cpm/ng; Ref.
23). The 150-bp DNA probe was
synthesized by 50 cycles of PCR (95 °C, 30 s; 55 °C, 30 s and 72
°C, 1 min) using 30 fmol of pKQV4 template and 10 pmol of each sequencing
oligo (defined above) in a 50-µl reaction mixture containing 40
mM Tris-HCl, pH 8.0, 5 mM MgCl2, 10
mM dithiothreitol, 50 mM NaCl, 50 µCi of
[
-32P]dCTP (6,000 Ci/mmol, Amersham Biosciences), 150
µM concentration each of the other three dNTPs, and 1 unit of
Taq DNA polymerase (Applied Biosystems).
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RESULTS
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Minigenes Harboring Codons from the Same Genetic Code Box Show Similar
ToxicityChanges in the last sense codon of some minigenes
determine variations in the degree of toxicity for pth mutants
(9,
24,
25). To understand the nature
of this effect, we used an expression library of two-codon ORF minigenes in
which the second, and last, sense codon was any of the 64 possibilities in the
genetic code (Fig.
1A). By maintaining the same transcriptional promoter, SD
region, initiation and termination codons, and the shortest possible
"ORF," we attempted to minimize the number of variables affecting
minigene expression and focus on the effect of the different sense codons on
toxicity (9,
13). The minigene constructs
were expressed in two pth mutants differing in the level of Pth
activitiy (pth[Ts] > pth[rap]; Ref.
15), as a means for ranking
minigene toxicities. The results, summarized in
Fig. 1B, showed a wide
variation in the degree of toxicity to the two mutant strains arising from
minigene expression and depending on the nature of the second codon: 27 were
toxic to both pth(Ts) and pth(rap), 18 were lethal to
pth(rap), and 16 had no deleterious effects on either mutant. Unlike
the toxic minigenes isolated by Tenson et al.
(24), none of our constructs
affected wild-type cell growth under the same assay conditions (data not
shown). This effect may be due to the lower strength of minigene expression in
the vector we used relative to that recorded for the vector used in the
previous report (24).
Minigenes with any of the three termination codons in the second position do
not encode peptides and are therefore expected to be harmless to Pth-defective
strains. In effect, the two constructs tested, carrying TAA and TGA
respectively, had no toxic effects (data not shown). No correlation was
observed between codon pair bias
(26) or cognate tRNA scarcity
(underlined codons in Fig.
1B; Ref.
22) and minigene toxicity. In
general, minigenes harboring codons grouped within the same genetic code box
(Fig. 1B) showed a
similar degree of toxicity (38 minigenes in 10 out of 16 boxes). Five of these
boxes contain codons for two different amino acids (e.g. TTN,
Phe/Leu; ATN, Ile/Met-, etc.), suggesting that the codons, rather than the
decoded amino acids, determine minigene toxicity. This would not be the case
for those minigenes bearing codons within each of the remaining six boxes in
which mixed degrees of toxicity were observed (dashed boxes in
Fig. 1B).
Non-toxic Minigenes Do Not Mediate Peptidyl-tRNA
AccumulationFor a number of minigenes, a direct correlation
between toxicity and peptidyl-tRNA concentration has been observed in
pth mutants (4,
9,
24). To assess the breadth of
this observation, we estimated the relative concentrations of peptidyl-tRNAs
for selected minigenes. Initially, we determined the time length of IPTG
minigene induction which generated the highest peptidyl-tRNA concentrations.
The data showed that, for the toxic (AAT and AAA) and partly toxic (GAT and
GAA) minigenes assayed, the highest accumulated fraction was reached in all
cases within a 30-min induction period
(Fig. 2B). Therefore,
all subsequent determinations of peptidyl-tRNA concentration were made at 30
min (Fig. 1B,
columns 4 and 5). The relative concentrations of peptidyl-tRNA
in C600pth(Ts) varied from 40 to 70% for toxic, 10 to 20% for partly
toxic, and 5 and 10% for non-toxic minigenes
(Fig. 1B, fourth
column in each cell). In C600rap, the relative concentrations of
peptidyl-tRNA from toxic and partly toxic minigenes were proportionally higher
(Fig. 1B, fifth
column in each cell), whereas the concentrations of peptidyl-tRNA for
non-toxic minigenes were lower, although similar to those observed for
pth(Ts). Wild-type transformants did not accumulate peptidyl-tRNA
(data not shown). Except for minigene AAT, encoding Asn, which intriguingly
promotes accumulation of peptidyl-tRNALys
(Fig. 2A),
heterologous peptidyl-tRNAs did not accumulate upon minigene expression:
minigene AAA did not accumulate peptidyl-tRNAAsn; minigene GAT did
not accumulate peptidyl-tRNAGlu; minigene GAA did not mediate
accumulation of peptidyl-tRNAAsp
(Fig. 2A); and
minigene AGA did not accumulate peptidyl-tRNALys.

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FIG. 2. Northern blot analysis and time course of peptidyl-tRNA accumulation
following expression of toxic and partly toxic minigenes. A,
Northen blot of peptidyl-tRNA (pep-tRNA) accumulation at different
times after IPTG induction of toxic, AAT and AAA, and partly toxic, GAT and
GAA, minigenes in E. coli C600rap. The peptidyl-tRNAs and tRNAs
(arrows), treated previously with CuSO4, were revealed by
complementary, 32P-labeled, oligos (see "Experimental
Procedures"). B, time course graph of percentage of
peptidyl-tRNAs accumulated in Northen blots shown in A.
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Peptidyl-tRNA Accumulation Depends upon the Specific Codon-tRNA
InteractionIn six of the genetic code boxes (dashed boxes
in Fig. 1B), minigenes
showed mixed degrees of toxicity. To understand the basis of these
differences, we analyzed peptidyl-tRNA accumulation upon minigene induction in
Pth-defective mutants. For example, in the set of minigenes carrying Arg
codons CGU, CGC, and CGA, for which tRNAArg-2 is the sole cognate
species, the generated peptidyl-tRNAs must be chemically identical. If we
assume an equal decoding rate for the mini-ORFs in each of the three
minigenes, toxicity would depend on the drop-off rate, because the rate of
peptidyl-tRNA hydrolysis by Pth is identical. The relative amounts of
peptidyl-tRNAArg-2 accumulated after expression of each minigene in
Pth-defective strains ranked in the order CGA>CGT>CGC, as expected from
their degree of toxicity (Fig.
3). Accordingly, the peptidyl-tRNA concentrations should reflect
the rate of peptidyl-tRNAArg-2 drop-off due to differences in
tRNAArg-2 interaction with each of the three codons (see
"Discussion").

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FIG. 3. Northern blot analysis of peptidyl-tRNAArg-2 accumulation and
tRNA species cognate to synonymous minigenes.
Peptidyl-tRNAArg-2 was determined after 30 min of IPTG minigene
induction in C600 (Wt), C600pth(Ts) (Ts), or
C600rap (rap) of toxic (CGA), partly toxic (CGT), and non-toxic (CGC)
minigenes. tRNA, aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA
(pep-tRNA) were detected as indicated in
Fig. 2 with (+) or without (-)
treatment with CuSO4 (see "Experimental Procedures").
The percentage peptidyl-tRNA accumulation in the presence of copper is given
at the bottom (same figures quoted in corresponding box of
Fig. 1B).
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tRNASer-1 is cognate to the codons in partly toxic minigenes TCT
and TCA and clearly accumulates as peptidyl-tRNASer-1 in strain
pth(rap) (40%, Fig.
1B); by contrast, accumulation of
peptidyl-tRNASer-5, produced by both the partly toxic TCT and the
non-toxic TCC minigenes, was not promoted in either strain
(Fig. 1B). This
suggests strongly that the toxicity of minigenes TCT and TCA is associated
with peptidyl-tRNASer-1 accumulation. The data for the minigenes
CTA and CTG could be explained in a similar way.
Non-toxic Minigene mRNAs Are TranslatedFor the expression
of different minigene variants, the cellular levels and stability of minigene
mRNA correlate with the strength of toxicity. It has been speculated that the
increased messenger stability results from a longer interaction period with
the ribosome during translation
(25). Results with Northern
blot assays confirmed that toxic minigenes (e.g. AAA, GTT, and CGA)
accumulated high levels of mRNA, whereas non-toxic minigenes (e.g.
GCC, GGC, and CGC) did not (data not shown). We then asked whether non-toxic
minigene mRNAs were translated at all and, if they were, why their expression
did not drive peptidyl-tRNA accumulation. Minigene AGA, lethal to
C600pth(Ts), and the non-lethal minigene GGC, were expressed in the
presence of antibiotics (Fig.
4). The antibiotics used were pactamycin, which causes ribosome
stalling soon after initiation of protein synthesis
(27), and erythromycin, which
enhances the dissociation of peptidyl-tRNAs containing at least six to eight
amino acids (6,
28). As expected, the
expression of the lethal minigene AGA resulted in mRNA accumulation even in
the absence of the antibiotics. The non-toxic GGC minigene, on the other hand,
accumulated mRNA only in the presence of pactamycin. Similar results were
obtained with the lethal CGA and the non-toxic CGC minigenes (data not shown).
These results indicate that mRNAs of these non-toxic minigenes are
translatable; furthermore, in the absence of antibiotics translation
termination should have occurred readily, suggesting that the mRNA-ribosome
complex may be short lived. Erythromycin did not mediate accumulation of
peptidyl-tRNA nor did it stabilize the ribosome-mRNA complex as deduced from
the observation that it did not favor mRNA accumulation
(Fig. 4). Given that the
erythromycin binding site is at the entrance of the 50 S ribosomal subunit
tunnel (29), it is not
expected to affect dipeptidyl-tRNAs which are too short to reach the site.

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FIG. 4. Northern blot analysis of mRNAs derived from minigenes with different
toxicity levels expressed in the presence of pactamycin and erythromycin.
Minigene mRNA accumulation was assayed in C600pth(Ts) cultures 30 min
after induction with IPTG. mRNAs (arrow) were revealed by probing
total RNA extracted from cultures of bacteria transformed by two minigenes
(AGA or GGC), in the presence of 50 µg/ml pactamycin (P) or
erythromycin (E) (C, no antibiotic treatment). The DNA probe
is described under "Experimental Procedures." The positions of
ribosomal RNA bands are also indicated.
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Efficient Translation Termination Prevents Peptidyl-tRNA
AccumulationTo test whether toxic and non-toxic minigenes differ
in the efficiency of translation termination, the levels of peptidyl-tRNA, the
intermediate previous to termination hydrolysis, were assayed under a
condition of defective termination (Fig.
5). An E. coli prfA1-pth(rap) double mutant,
defective for both RF1 (thermosensible) and Pth activities, was transformed
with constructs carrying minigenes causing different degrees of lethality. In
mutant prfA1 the activity of RF1 is greatly reduced at 43 °C
(16). We used the partly toxic
CGT minigene and the non-toxic variant CGC, for which tRNAArg-2 is
the sole cognate isoacceptor. When RF1 was defective, expression at 32 °C
of the CGC minigene did not promote detectable peptidyl-tRNAArg-2
accumulation, whereas it did at 43 °C (lane 4 versus lane 8). The
partly toxic CGT minigene promoted peptidyl-tRNAArg-2 accumulation
at both temperatures (lanes 2 and 6), and accumulation was
enhanced at 43 °C. These results suggest that efficient translation
termination of non-toxic minigene mRNAs shortens peptidyl-tRNA residence time
on the ribosome so that drop-off events remain undetectable. In strain
prfA1, which harbors the wild-type pth allele (lanes
10 and 12), no peptidyl-tRNA was identified during toxic
minigene expression at 43 °C; this suggests that all the generated
peptidyl-tRNA eventually dissociates from the ribosomes and is cleaved by Pth
in solution.

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FIG. 5. Northern blot assays of peptidyl-tRNAArg-2 accumulation
during synonymous minigene expression under limiting RF1 and Pth
activities. IPTG induction of CGT and CGC minigene expression in E.
coli strains US486 [prfA1] and GG06 [pth(rap)
prfA1] was done for 30 min at 32 or 43 °C. tRNA species were
treated, with (+) or without (-) treatment with CuSO4, and revealed
as indicated in Fig. 3.
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DISCUSSION
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We used a library of two-codon minigenes to analyze the effect of the
variable second codon on Pth-defective cells. The minigenes were ranked as
fully toxic, partly toxic, and nontoxic. All analyzed minigenes showed that
these categories correlated directly with relative levels of the respective
peptidyl-tRNA concentrations. With few exceptions, the four minigenes bearing
codons from the same genetic code box showed similar degrees of toxicity
(Fig. 1B). This rule
held for unmixed boxes, i.e. those encoding only one type of amino
acid, as well as for mixed boxes, encoding two amino acids. A nucleotide
sequence relationship has been reported for the tRNAs cognate to the codons
within the same genetic code box
(30,
31). It is, therefore, likely
that the chemical nature of the cognate tRNA is an important toxicity
determinant. A closer look at the exceptions mentioned above seems to confirm
this conclusion.
A long ribosome pause on the last sense codon, arising from inefficient
translation termination, eventually results in peptidyl-tRNA dissociation and
mRNA stabilization (9,
12). Therefore, toxic
minigenes would be defective in translation termination, and non-toxic
minigenes would terminate readily. Additional support for this idea comes from
two types of evidence: first, mRNA of the non-toxic CGC minigene was
stabilized by pactamycin (data not shown), an antibiotic which arrests protein
synthesis early after initiation and results in the accumulation of a 40
S·Met-tRNAF·mRNA complex
(32); second, the expression
of the same CGC minigene promoted the accumulation of normally undetectable
peptidyl-tRNA under limited activity of the termination factor RF1
(Fig. 5, lanes 7 and
8; Ref. 16). We were
unable to find a direct correlation between the minigene mRNA and
peptidyl-tRNA accumulation, i.e. the length of the ribosomal pause
and the strength of the codon:anticodon bond. If this strength has an
influence on the ribosomal pause, it might be part of a more complex
interaction which involves several factors. One factor could be the effect of
both the amino acid side chains and the tRNA body of the peptidyl-tRNA on the
interaction with the ribosome, in a manner similar to their themodynamic
contribution to the binding of the aminoacyl-tRNA and the EF-Tu
(33). A second factor
affecting ribosomal pausing may be the specific architecture of the
codon:anticodon interaction. The Arg codons CGA, CGU, and CGC are decoded by
the unique tRNAArg-2
(22). Unlike CGC, CGA is a
poor 5' context for amber suppression by the su7-encoded tRNA suggesting
that the CGA codon-peptidyl-tRNAArg-2 in the ribosomal P-site can
interfere with either the translation of a codon in the A site
(34) or with enhanced
termination at a stop codon. We showed that minigene CGA clearly promoted
peptidyl-tRNA accumulation, whereas minigene CGC did not (Figs.
1B and
3), thus indicating that CGA
hinders termination. It has been speculated that the A:I pairing of the CGA
codon with the tRNAArg-2 anticodon at the wobble position is
structurally distorted, thus hampering decoding
(34,
35). Our results, however,
showed that synthesis of peptidyl-tRNA occurs readily
(Fig. 3), implying that the
defect may be at mRNA translation termination. It is possible that wobble
position base pair distortion affects translation termination, because
minigene CGT, which accumulates intermediate levels of peptidyl-tRNA
(Fig. 3), has a less severe
distortion at the U:I pair in the CGU-anticodon interaction
(34). A similar mechanism
could apply for minigenes AGT and AGC, for which tRNASer-3 is the
cognate species.
A third factor affecting ribosomal pausing could be the interaction between
tRNA in the ribosomal P-site and the release factor in the A-site. For
example, in the presence of peptidyl-tRNAGly-3 in the P-site at
codons GGA/G, termination efficiency at UAG is higher than in the presence of
peptidyl-tRNAGly-2 at the same codons, suggesting an unusual
interaction between tRNAGly-2 and RF1
(36). This hypothesis could
explain why toxicity and peptidyl-tRNA accumulation for TCT and CTG in the
corresponding minigenes is associated to only one of the two cognate tRNAs
(Fig. 1B).
What is the distribution of "toxic" and "non-toxic
codons" in bacterial genes? Interestingly, AAA/G and AAU/AAC codons in
minigenes, which promote high rates of peptidyl-tRNA accumulation
(Fig. 2), are frequently
located at the beginning of E. coli ORFs. These codons enhance
efficiency of translation when substituted at positions two and three of a
reporter gene (37,
38). By contrast, codons that
promoted low rates of peptidyl-tRNA accumulation are rarely located among the
first three positions (e.g. CUN, GCN, and GGN gene code boxes; Refs.
37 and
38). In the pth(ts)
mutant, families of tRNAs cognate to these codons are among those with the
lowest rates of accumulation of the corresponding peptidyl-tRNAs at
non-permissive temperatures
(11). The presence of codons
prone to drop-off in minigenes may represent an advantage for protein
synthesis when located at the initial positions in the mRNA ORFs. These codons
might act as sensors of the general availability of charged tRNAs. If the
availability is appropriate, elongation proceeds, otherwise abortive drop-off
occurs. This strategy would prevent wasteful protein synthesis elongation
under limiting tRNA availability.
Interestingly, a high frequency of AAA/G codons, associated to high
drop-off rates in minigenes, has been found at the last sense position of the
E. coli ORFs where they also promote drop-off
(13). This non-random codon
distribution at the ends of ORFs has been considered as an important factor in
the modulation of translation termination
(39). This could be explained,
because such codons provide an alternative translation termination mechanism
including drop-off and Pth-mediated hydrolysis of the final peptidyl-tRNA.
 |
FOOTNOTES
|
---|
* This work was supported by Consejo Nacional de Ciencia y Tecnología
(CONACyT) Grants 28401N and 37759N (to G. G.) and O28 (ER025) (to Dr. Julio
Collado Vives (Universidad Nacional Autónoma de México)) and by
the Consejo del Sistema Nacional de Educacién Tecnológica
(COSNET). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Supported by a postdoctoral fellowship from COSNET and funds from CONACyT
Grant O28. 
During this time was a Université de Paris VII visiting professor and
was awarded a sabbatical fellowship by CONACyT. To whom correspondence should
be addressed. Tel.: 52-55-747-3338; Fax: 52-55-747-7100; E-mail:
guarnero{at}lambda.gene.cinvestav.mx.
1 The abbreviations used are: Pth, peptidyl-tRNA hydrolase; IPTG,
isopropyl-1-thio-
-D-galactopyranoside; ORF, open reading
frame; oligo, oligodeoxyribonucleotide; SD, Shine-Dalgarno sequence. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Marco A. Magos Castro, José Bueno Martínez, and
Guadalupe Aguilar González for skilful technical support; Benito
Aguilar for obtention of the random minigene constructs; Richard Buckingham
for kindly providing strain US486; Jon Gallant for his comments on the
manuscript; and Philippe Régnier and associates at The Institut de
Biologie Physico-Chimique for the generous hospitality, which made possible
writing early versions of this paper.
 |
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