Evidence that the bypassing ribosome travels through the coding gap.

Abstract

In translational bypassing, a peptidyl-tRNA::ribosome complex skips over a number of nucleotides in a messenger sequence and resumes protein chain elongation after a "landing site" downstream of the bypassed region. The present experiments demonstrate that the complex "scans" processively through the bypassed region. This conclusion rests on three observations. (i) When two potential "landing sites" are present, the protein sequence of the product shows that virtually all ribosomes land at the first and virtually none at the second. (ii) In such a sequence with two landing sites, the presence of a terminator triplet in phase in the coding region immediately after the first landing site drastically reduces the efficiency of bypassing. (iii) Internally complementary sequences that can form a stable stemloop in the bypassed region significantly reduce the efficiency of bypassing. We analyze bypassing from a given "takeoff" site to "landing sites" at different distances downstream so as to derive estimates of the frequency of ribosome takeoff and of the stability of the bypassing complex.

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Evidence that the bypassing ribosome travels through
the coding gap
Jonathan Gallant*, Paul Bonthuis, and Dale Lindsley
Department of Genome Sciences, University of Washington, P.O. Box 357730, Seattle, WA 98105
Edited by Charles Yanofsky, Stanford University, Stanford, CA, and approved August 29, 2003 (received for review June 17, 2003)
In translational bypassing, a peptidyl-tRNA::ribosome complex
skips over a number of nucleotides in a messenger sequence and
resumes protein chain elongation after a ‘‘landing site’’ down-
stream of the bypassed region. The present experiments demon-
strate that the complex ‘‘scans’’ processively through the bypassed
region. This conclusion rests on three observations. (i) When two
potential ‘‘landing sites’’ are present, the protein sequence of the
product shows that virtually all ribosomes land at the first and
virtually none at the second. (ii) In such a sequence with two
landing sites, the presence of a terminator triplet in phase in the
coding region immediately after the first landing site drastically
reduces the efficiency of bypassing. (iii) Internally complementary
sequences that can form a stable stem?loop in the bypassed region
significantly reduce the efficiency of bypassing. We analyze by-
passing from a given ‘‘takeoff’’ site to ‘‘landing sites’’ at different
distances downstream so as to derive estimates of the frequency
of ribosome takeoff and of the stability of the bypassing complex.
I
mation from two noncontiguous ORFs into the sequence of a
single, continuous polypeptide chain (reviewed in ref. 1). The
prototypical case has been the translation of the phage T4 gene
60 mRNA coding for a topoisomerase subunit, which involves
the bypassing of a 50-nt untranslated segment (or ‘‘coding gap’’)
between a GGA ‘‘takeoff’’ triplet and a GGA ‘‘landing site’’
triplet. (In the text, we use boldface to show RNA triplets and
normal type for triplets or other sequences in DNA.) This case
of bypassing depends on very specific features of the sequence
(1–3):(i)identityofthetakeoffandlandingsitetriplets,implying
that peptidyl-tRNA is what makes the trip; (ii) optimum spacing
of the takeoff and landing sites; (iii) a terminator triplet imme-
diately 3? of the takeoff triplet; (iv) a stem-loop of optimal
stability and placement in the region after the takeoff site; and
(v) a particular amino acid sequence of the peptide encoded in
a region 5? of the takeoff site. The role of these sequence
elements and of participating factors has been investigated in
detail (1, 4–6).
The sequence rules governing this kind of bypass event seem
very constraining. We have reported on a type of bypass event
that seems to be subject to more relaxed rules and may therefore
occur on a wide variety of sequences. This is a bypass induced by
ribosome pausing at a ‘‘hungry’’ codon calling for an aminoacyl-
tRNA in short supply (7, 8). We showed that this kind of bypass
shared with the gene 60 case only the requirement for synony-
mous takeoff and landing sites. Otherwise, it could be demon-
strated over various coding gap distances (from 7 to at least 40
nt), and seemed largely independent of the sequence in the
coding gap and upstream of it (7). In those experiments and the
present ones, we inhibited the activation of isoleucine with
isoleucine-hydroxamate (ILHX), thus inducing ribosomes to
pause at a hungry AUA codon immediately following a UUC
takeoff site. Protein sequencing of the product established the
occurrence of bypassing from the takeoff site to synonymous
landing sites located at various downstream positions (7).
The data reported previously demonstrate that the
ribosome::peptidyl-tRNA complex departs from the takeoff
n ‘‘translational bypassing,’’ a ribosome skips over a number of
nucleotides in a messenger sequence so as to join the infor-
codon and resumes protein chain elongation immediately after
the synonymous landing site codon. It seems intuitively likely
that the complex slides through the intervening or coding gap
region while remaining associated with the mRNA and influ-
enced by elements in its sequence, a model Herr et al. (1) refer
to as ‘‘scanning.’’ However, it remains conceivable that the
ribosome::peptidyl-tRNA complex detaches completely from
the message and somehow hops over the coding gap to reengage
at the landing site, a theoretical possibility that the authors of the
aforementioned review term ‘‘bridging.’’
In the present communication, we report on experiments
designed to distinguish between these two formal models by
showing that the ribosome::peptidyl-tRNA complex can interact
with elements in the bypassed region and that it encounters these
elements sequentially along the length of the coding gap. Our
results demonstrate that the complex does scan through the
coding gap sequence, although in a mode that precludes normal
translation. We have also examined the effect near the landing
site of the tetranucleotide AGCT, hypothesized to be a frame-
keeping motif (9), and found that it has no effect on bypassing
efficiencyinourexample.Furtherconsiderationofthebypassing
phenomenon leads to the hypothesis that codon::anticodon
matching in the P-site strongly influences the ability of the
adjacent A-site to accept aminoacyl-tRNA.
Materials and Methods
All bypass reporter constructs were made by force-cloning
appropriate oligonucleotides with HindIII and BamHI over-
hangs into plasmid pEK-0F, as described (7). Reporter plasmids
were transfected into host strain C92 (relA spoT) carrying the O6
deletion of the early part of lacZ (10), and selected and analyzed
as described (7, 11). To confirm the engineered lacZ sequences
of the constructs, ?600-bp PCR products were amplified (using
primers flanking the oligonucleotide insert) and sequenced at
the University of Washington Biochemistry DNA Sequencing
Facility. Cultivation of cells (M63-glucose minimal medium,
37°C, forced aeration) was as described except that there were no
amino acid supplements. The lac promoter was induced in
log-phase cultures at an OD690of ?0.2 by the addition of 2 mM
isopropyl-?-D-thiogalactopyranoside (IPTG) and 2.5 mM
cAMP,andsubculturesweregrowninparallelintheabsenceand
presence of 72 ?g?ml ILHX to inhibit isoleucine-tRNA charg-
ing. In all experiments, the glucose concentration was 0.07%,
which limits growth in ILHX to about one doubling (an increase
of ?40 ?g?ml protein from the point at which we induce);
inhibited cultures were followed long enough to confirm growth
inhibition, typically 3-fold, and then allowed to continue bub-
bling overnight, during which glucose was exhausted and growth
ceased, and the cultures were harvested the next morning.
Samples were processed for enzyme and protein assay, and for
?-galactosidasepurificationandaminoacidsequenceanalysis,as
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: ILHX, isoleucine-hydroxamate.
*To whom correspondence should be addressed. E-mail: jgallant@u.washington.edu.
© 2003 by The National Academy of Sciences of the USA
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described by Barak et al. (11). Enzyme and protein assays, and
calculations, were as described by Lindsley et al. (8). One enzyme
unit corresponds to the quantity of enzyme that yields a rate of
change of optical density at 420 nm of 1.0 per minute under the
specified assay conditions; milli-enzyme units are presented for
convenience. Predicted secondary structures in mRNA, and
their stabilities, were generated by RNASTRUCTURE Version 3.71
software (12).
Results
Bypassing on Sequences with Two In-Frame Landing Sites. To dis-
criminate between ‘‘scanning’’ and ‘‘bridging,’’ we have asked
how the bypassing process responds to certain kinds of sequence
information within the coding gap. In the first such test, we
examined starvation-inducible bypassing on a sequence with two
potentially productive landing sites. Fig. 1 shows that bypassing
from a TTC takeoff site occurs at similar frequencies whether a
single TTC landing site is located 10 or 16 nt downstream,
corresponding to 3–5% of the lacZ? control. To determine
whether the ribosome::peptidyl-tRNA complex passes through
position10onthewaytoposition16,wemadethethirdconstruct
listed in Fig. 1A, possessing both of these landing sites. Starva-
tion-induced bypassing on this construct occurred with an effi-
ciency (6.4%) that was slightly higher than, but in fact not
statistically distinguishable from, that on the construct with a
single landing site 10 nt after the takeoff site (Fig. 1). To
determine which landing sites were used when these two were
available, we determined the amino acid sequence of the protein
generated through starvation-induced bypassing.
If the complex hops or ‘‘bridges’’ to independent landing sites,
then the resulting protein should be a mixture of two species,
reflecting independent landings, some at position 10 and some
at position 16. The protein sequence signal in that case would be
uniform up to the F at the takeoff site, but, after that, it would
show two predominant species, corresponding to the two landing
sites. However, if the complex slides or scans through the
bypassed region, then it must reach position 10 first. If the
efficiency of landing is high, then the protein sequence should
resume uniformly at the codon following that at position 10. The
protein sequence data shown in Fig. 2 unequivocally favor the
second interpretation. A secondary signal of protein that re-
sumed chain elongation at position 16 would be detectable if it
represented as much as 10% of the total, and no such signal is
apparent. We conclude that essentially all of the ribosomes
resume protein chain elongation at position 10 rather than
position 16, as they would naturally do if they moved through the
coding gap and therefore always encountered position 10 first.
This interpretation predicts that a terminator codon placed
after the landing site at position 10 should abolish bypassing in
a two-landing-site construct. In fact, bypassing on this construct
(the fourth entry in Fig. 1A) was not quite abolished altogether,
but it was reduced from 6.4% to 0.88%. This large reduction
supports the conclusion from protein sequencing that the great
majority of ribosomes resume chain elongation at the first of the
two landing triplets. The small residual activity suggests that a
small minority of ribosomes, of the order of 14%, either can pass
through a landing site triplet (without resuming chain elongation
and then land at a more distal landing triplet) or can hop or
bridge directly from the takeoff site to the more distal landing
site. A third possibility is that all of the ribosome::peptidyl-tRNA
complexes did land at position 10, but that the immediately
following terminator induced 14% of them to bypass a second
time to the distal landing site. The protein sequence of the
residual enzyme produced in construct TTC–10–STOP–16–
TTC was determined (not shown); it lacked any of the amino
acids encoded between the takeoff site and the distal landing site
at position 16, which is consistent with any of these three
explanations.
To test the generality of preferential landing at the first of two
landing sites, we also constructed TTC–16–22–TTC (Fig. 1), and
purified the ?-galactosidase produced during ILHX-inhibited
growth. The amino acid sequence of this protein (not shown)
Fig. 1.
vectortomakeeachconstructanditsrateofreporter?-galactosidasesynthesis.Theoligonucleotideinserts(shownascodingstrandonly)wereligatedbetween
the HindIII and BamHI sites, replacing a 20-mer of the pEK-0f vector, as described in Materials and Methods. The TTC takeoff and landing site triplets are shown
in bold type, with the latter underlined; the hungry ata codon (coding for isoleucyl-tRNA) is shown in lowercase; a dash is used as a spacer between contiguous
nucleotides in the lacZ? control construct, to keep all sequences aligned. ILHX refers to subcultures subjected to 72 ?g?ml ILHX to partly inhibit the attachment
of isoleucine to its cognate tRNAs. ?-Galactosidase synthesis was measured over about one mass doubling after induction. It is expressed as induced specific
activity or increase in ?-galactosidase activity divided by increase in protein, in milli-enzyme units?mg protein; the averages of the number of replicate
experiments shown in parentheses, ? SEMs, are tabulated. [The minor difference in ILHX-stimulated ?-galactosidase between TTC–10–TTC (13,900) and
TTC–10–16–TTC(18,800)isnotsignificant(t?1.33,P?0.20).]The%bypassingistheinducedspecificactivityofthebypassreporterdividedbythecorresponding
valueinthelacZ?construct(TTC–9–15–TTClacZ?),whichhasasequencecloselysimilartothebypassreportersbutcodesin-framefor?-galactosidase.(B)Shown
is the relevant region of the vector’s lacZ gene (coding strand), with lines from A indicating the insertion of the oligonucleotides into the cloning sites. The
translationproductshownbelowhastheenterokinase(EK)recognitionsequenceunderlinedandthecleavagesitemarkedbyanarrow.Thetranslationproduct
oftheinsertedoligo(boldface)alwaysincludesS-Y-F,thenavariablesequenceofnaminoacidssymbolizedas( )n,andthenN,L,andaGencodedattheBamHI
site.
Bypassing efficiencies of constructs with one or two in-frame landing sites. (A) Shown are the oligonucleotide inserts cloned into the lacZ gene of the
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demonstrated that essentially 100% of the landings were at
position 16, and again no secondary signal of landings at the
second site, that at position 22, were detectable.
The Effect of Secondary Structure in the Coding Gap. If the
ribosome::peptidyl-tRNA does scan through the bypass region,
its progress might be affected by noncoding structural features
of the region’s sequence, such as a stable stem?loop. We
therefore compared the efficiency of bypassing in the set of
constructs shown in Fig. 3. One, termed SL-39, has internally
complementary sequences in the 39-nt coding gap so as to form
a very stable stem?loop (?24 kcal, according to the RNASTRUC-
TURE program). A second construct, NSL-39, differs from SL-39
at seven positions in the downstream arm of the stem so that no
stable secondary structure can be formed. Next, we changed the
upstream part of the stem to nucleotides complementary to
those in the downstream arm region of NSL-39 so as to generate
a new stem?loop construct named SL2–39; this construct forms
a stem?loop(?16 kcal) in the same position as SL39, but of very
different sequence. For bypass constructs SL-39 and NSL-39, we
made corresponding lacZ? controls, as shown in Fig. 3. Finally,
we indicate the sequence of L-39, on which we reported earlier
(7). In this construct, there is also no expected secondary
structure, but the coding gap sequence is very different from that
of NSL-39. Fig. 3C reports enzyme synthesis in all these con-
structs, both the very low values during normal growth and the
much higher values during isoleucyl-tRNA limitation to stimu-
late bypassing at the hungry AUA codon.
It can be seen that starvation-induced enzyme synthesis in
both SL-39 and SL2–39 was ?3.0 times lower than it was in
NSL-39, a highly significant difference (see the statistics quoted
in the legend to Fig. 3). However, in the corresponding lacZ?
controls, there is no significant difference between the SL and
the NSL constructs. It follows that the stem?loop sequence does
not detectably affect lacZ expression or normal translation.
Rather, the effect on enzyme synthesis encoded by the bypass
reporters must reflect an effect on the bypassing process spe-
cifically. Fig. 3B displays the bypassing efficiencies, defined as
enzyme synthesis in a bypass construct divided by that in the
corresponding lacZ? construct under the same conditions. Note
the large difference between the starvation-induced bypassing
efficiencies of both stem?loop constructs, on the one hand, and
NSL39, on the other.
Weconcludethatproductivebypassingismarkedlyreducedby
some aspect of the sequence difference between both of the SL
constructs and NSL-39. The difference from NSL-39 that both
SL constructs share is, of course, the presence of a stem?loop.
Theidentical,low-bypassingefficienciesofthetwoSLconstructs
suggest that the existence of a stem?loop, rather than its precise
sequence, is what matters.
Other features of the coding gap sequence seem much less
relevant. The two constructs without a stem?loop, NSL39 and
L39, differ from each other at 22 of 39 positions in the coding
gap. Nonetheless, they both display high bypassing efficiencies,
not statistically distinguishable from one another, but much
higher than that of the two SL constructs which contain a
stem?loop.
Is AGCT a Frame-Keeping Marker? Henaut et al. (9), on the basis of
genomic survey information, have suggested that the tetranucle-
otide motif AGCT might be a framekeeping signal in translation.
Our system provides one way to test this proposal. The sequence
on which we first demonstrated a highly efficient, 16-nt bypass
had, as it happens, an AGCT in the coding gap, 2 nt before the
landing site. To test whether the motif plays any role in correct
landing, we have made another construct in which the AGCT
Fig. 2.
construct TTC–10–16–TTC was subjected to ILHX inhibition for approximately one doubling. The ?-galactosidase was purified, cut with enterokinase (EK),
separated by PAGE, and blotted to poly(vinylidene difluoride) membrane as described (7). The blotted protein was subjected to automated Edman sequence
analysis at the Molecular Genetics Instrumentation Facility of the University of Georgia. Shown is the nucleotide sequence of the DNA-coding strand starting
at the 3? (or C-terminal) side of the region encoding the EK cleavage site. The hungry ata codon, subject to aminoacyl-tRNA limitation, is shown in lowercase;
there are no other ata codons in the lacZ-coding sequence. The TTC takeoff site and both TTC landing sites are shown in bold type, with the latter underlined;
and the terminator blocking the initial reading frame is overlaid by a hexagonal ‘‘stop’’ sign. The amino acid encoded is specified in parentheses above each
relevant triplet. The cycle bar plot presents lag-corrected pmols, summed from three sequencing runs on two independent blots. Each cycle of the cycle bar plot
is aligned over the triplet encoding that cycle’s amino acid signal, with a dashed arrow over the coding gap. The amino acid signal begins with the valine residue
on the C-terminal side of the EK cleavage site.
Amino acid sequence of the bypass region of ?-galactosidase encoded by a bypass reporter with two landing sites. A 2-liter culture of cells harboring
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tetranucleotide was replaced by GAGT. As Table 1 shows, the
bypassing efficiency on the two sequences showed no significant
difference. We made another pair of constructs in which the
takeoff?landing site pair are TAC, one of which has AGCT 2 nt
upstream of the landing site while the other has GAGT at the
same position. These two constructs also showed identical
bypassing efficiency (Table 1).
Discussion
Our findings argue that the ribosome::peptidyl-tRNA complex
gets from the takeoff site to the landing site by scanning
processively through the untranslated coding gap. When two
productive, landing triplets in frame with the reporter lacZ
sequence are available, the protein sequence shows that all of the
ribosomes resume protein chain elongation at the first landing
triplet and virtually none at the second; this finding argues that
no ribosomes can reach the second landing triplet that have not
already interacted with the first, i.e., processivity. The finding
also indicates that very few ribosomes scan past a productive
landing site. Precisely the same conclusions follow from the
much diminished bypassing we detected in a two-landing site
construct with a terminator codon after the first landing site. It
Fig.3.
to display predicted mRNA secondary structures. The takeoff and landing UUC codons are shown in bold type, with the latter underlined; the hungry aua codon
is in lowercase. In the sequence of each construct after SL-39, bases that differ from SL-39 are in italic type. (B) Displayed are the percent bypassing, defined as
theinducedspecificactivityofthebypassreporterunderthespecifiedconditiondividedbythatofthecorrespondinglacZ?.Filledbars,unstarvedcontrols;gray
bars, ILHX inhibited. (C) The mean induced specific activities of ?-galactosidase are as in Fig. 1 for each case analyzed, with SEM; the number of replicates is in
parentheses. The significance of the differences between the ILHX-specific activities was evaluated by t test. For the difference between SL-39 and NSL-39,
t ? ?4.7 and P ? 0.005; for the difference between SL2–39 and NSL-39, t ? ?4.2 and P ? 0.002. The modest difference between the two constructs with no
stem?looop, L-39 and NSL-39, is not significant: t ? 1.795 and P ? 0.10.
Theeffectofsecondarystructureonbypassing.(A)ShownistheRNAtranscriptoftheinsertedoligonucleotideportionofthelacZgeneofeachconstruct,
Table 1. Activity of bypass constructs with and without AGCT tetranucleotide
ConstructInserted oligonucleotide
?-Galactosidase synthesis
Unstarved ILHX
L-16 (? AGCT)
L-16 (no AGCT)
TAC–16–TAC (? AGCT)
TAC–16–TAC (no AGCT)
5?-AGCTATTTCataATCT [AGCT] AATTTGAAGATAATTTAGG-3?
5?-AGCTATTTCataATCT [GAGT] AATTTGAAGATAATTTAGG-3?
5?-AGCTATTACataATCT [AGCT] AATACGAAGATAATTTAGG-3?
5?-AGCTATTACataATTT [GAGT] GATACGAAGATAATTTAGG-3?
214 ? 15 (4)
278 ? 31 (4)
348 ? 34 (15)
272 ? 23 (5)
17,400 ? 1,280 (4)
27,200 ? 4,460 (4)
21,800 ? 4,510 (10)
20,700 ? 2,690 (4)
The bypass sequences shown are the coding strands of the oligonucleotides. The TTC takeoff and TTT landing sites are shown in boldface, with the latter
underlined, and the hungry ata codon is in lowercase; the AGCT tetranucleotide and the GAGT replacing it are bracketed. Induced specific activies of
?-galactosidase are as in Fig. 1. ?-Galactosidase synthesis is expressed as induced specific activity or increase in ?-galactosidase activity divided by increase in
protein, in milli-enzyme units?mg protein; the averages of the number of replicate experiments shown in parentheses, ? SEMs, are tabulated.
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showed an 86% reduction in bypassing, suggesting that the
efficiency of landing at the first, nonproductive landing site was
0.86 among scanning ribosomes. However, the residual 14%
activity measured in this construct did not show up as a 14%
secondary signal in the protein sequence data (Fig. 2), which, of
course, comes from a two-landing-site construct without the
terminator. We speculate that the presence of a terminator
triplet immediately after the first landing site stimulated an
additional hop to the second landing site at a low frequency,
similar to the original description of low-level bypassing by Weiss
et al. (13). In any case, this is a minor discrepancy, and the strong
conclusion from both experiments remains that very few ribo-
somes go past a potential landing site to reach a second one. In
other words, once a scanning peptidyl-tRNA::ribosome complex
reaches a cognate landing site, the probability that it will resume
protein chain elongation there rather than continue scanning is
at at least 0.86 and probably closer to 1.0.
Let us consider the probabilities that enable bypassing over a
distance of n nucleotides. First, a ribosome::peptidyl-tRNA
complex must disengage from the messenger at the takeoff site;
call that probability PT. Once a complex has disengaged, it must
survive in a state capable of resuming protein chain elongation
as it moves through n nucleotides; call that probability PS,n.
Finally, the scanning ribosome must resume chain elongation
(i.e., land) at the specified landing site rather than continue
scanning, probability PL. Thus, the joint probability of successful
bypassing from a given takeoff site to a given landing site n
nucleotides downstream is
PB,n? PT?PS,n?PL.
[1]
As we have seen above, PLis close to 1.0 for the present case.
We find that PB,ndecreases with n, reflecting the instability of
the scanning complex. Let us make the simplest assumption that
the inactivation of the complex as it traverse n nucleotides of
coding gap is a first-order decay process with a decay constant
proportional to the number of nucleotides. (This formulation is
equivalent to assuming a random inactivation with a probability
proportional to the number of nucleotides.) In other words,
PS,n? e?kn. Therefore,
PB,n? PT?e?kn.
[2]
Because all our constructs have precisely the same takeoff
sequence, they must share the same value of PT. As a result of
this identity, we can estimate both PTand k from the decline in
PB,nin our constructs with different values of n.
PB,nfor L–39 (in which we earlier demonstrated the bypass
through direct amino acid sequencing) is 0.0057 (Fig. 3). PB,nfor
TTC–10–16–TTC (in which the present sequence data demon-
strate that all landings were at position 10) is 0.064 (Fig. 1).
Therefore,
0.0057 ? PT?e?39k
and 0.064 ? PT?e?10k.
[3]
SolvingthetwosimultaneousequationsyieldsPT?0.15,andk?
0.084. Thus, our ILHX inhibition regime induces ?15% of the
peptidyl-tRNA::ribosome complexes that encounter the hungry
codontotakeoff,and,astheymovethroughthecodinggap,they
are inactivated at an average rate of about one hit per 12 nt
moved. In the case of the background, unstarved bypassing, we
do not have direct protein sequence data to confirm that enzyme
activity reflects the expected bypass event. Nonetheless, assum-
ing that it does, we can solve the corresponding pair of simul-
taneous equations for the background values of PB, which are
0.000045 and 0.00058, respectively. The solutions are PT ?
0.0014 and k ? 0.088. Thus, ILHX inhibition increases the
probability of takeoff 100-fold, but, as one would expect, has no
effect on k.
The inactivating hits subsumed in the PS,n (or e?kn)term
comprise all events that disqualify the complex from resuming
protein chain elongation at a potential landing site. We assume
that most hits are simple dissociation of the peptidyl-tRNA from
the ribosome; the continuous generation of peptidyl-tRNA
(detected after thermal inactivation of peptidyl-hydrolase) has
long been known to occur (14). An unknown proportion of the
hits may be scavenging of the peptidyl-tRNA::ribosome complex
by the SsrA-RNA system (refs. 15 and 16, and reviewed in
ref. 17) whereas another unknown proportion undoubtedly
reflects unproductive landings at noncognate, out-of-frame
landing sites (refs. 1 and 5, and J.G. and D.L., unpublished
experiments). Menninger (14) has estimated the rates of disso-
ciation of various peptidyl-tRNAs during translation as lying
between 0.00013 and 0.004 per elongation cycle. Our estimate
that the peptidyl-tRNA::ribosome complex decays with a prob-
abilityof?0.084pernucleotidemovedimpliesthatthebypassing
complex is much less stable than the translating ribosome.
What determines the stability of the bypassing complex? The
experiments of Fig. 3 demonstrate that secondary structure in
the coding gap has a systematic effect. On the other hand,
sequence differences other than secondary structure seem to
have little or no effect. There is no difference in bypassing
efficiency between SL-39 and SL2–39, which have stems of
different sequence; nor is there much difference between the
high-bypassing efficiencies of NSL-39 and L-39, which have very
different sequences in the coding gap, but no secondary struc-
ture. In our previous report (7), we found identical bypassing
efficiencies over a 22-nt coding gap in two constructs that
differed at six positions in the gap. In other experiments, we have
found identical bypassing efficiencies over a 16-nt coding gap in
constructs differing by as many as five positions (unpublished
experiments). Taken together, these comparisons strongly sug-
gest that primary sequence in the bypass region has little effect
on the efficiency of bypassing (and therefore the stability of the
bypassing complex) unless it permits a secondary structure to
form.
Most likely, stem?loop structures simply decrease the rate of
ribosome movement through the coding gap, thereby allowing
more time for the peptidyl-tRNA::ribosome complex to disso-
ciate. We might note that the same stem?loop that diminished
bypassing had no effect on normal lacZ expression and enzyme
synthesis from a lacZ? control sequence. Thus, if the translating
ribosome is slowed down by secondary structure while traversing
this region of this message, the decrease is too small relative to
the transit time for the entire coding sequence to affect protein
yield significantly. This observation comes as no surprise, inas-
much as the 39-nt region in question represents ?1.5% of the
entire length of the lacZ-coding sequence. In a bypass reporter,
in contrast, the 39-nt distance becomes critical precisely because
of the instability of the bypassing complex: it must get through
the coding gap before peptidyl-tRNA dissociates (or SsrA RNA
is recruited) to resume translation at the landing site.
The present results seem to differ strikingly from the T4 gene
60 system, wherein a stem?loop structure stimulates rather than
diminishesbypassing.Inthegene60case,however,thebypassing
signalmustincludeaterminatorsequence(UAGC)thatispoorly
recognized by release factor 1, as well as the stem?loop located
immediately 3?-ward. These two elements may have their joint
effect by forcing the ribosome to pause at the UAG without
binding a release factor (1). In our experiments, we dictate a
ribosome pause at the hungry AUA codon by limiting the
charging of cognate isoleucyl-tRNA. A ribosome pause dictated
by limiting the availability of a specific tRNA isoacceptor
stimulates bypassing as well (8), presumably by the same mech-
anism. We have elsewhere shown that bypassing can be stimu-
lated not only at a hungry AUA codon, but also at other hungry
codons, including AUC (unpublished experiments), AAG (ref. 7
13434 ?
www.pnas.org?cgi?doi?10.1073?pnas.2233745100Gallant et al.