Article

Heterogeneity of Stop Codon Readthrough in Single

Bacterial Cells and Implications for Population
Fitness
Graphical Abstract Authors
Yongqiang Fan, Christopher R. Evans,
Karl W. Barber, ..., Oleg A. Igoshin,
Jesse Rinehart, Jiqiang Ling

Correspondence
jiqiang.ling@uth.tmc.edu

In Brief
Protein synthesis accuracy is important
for cell physiology and has been mostly
studied at the population level. Fan et al.
develop a dual-reporter system to
quantitate protein synthesis errors in
single bacterial cells and show that
slowing protein synthesis increases some
translational errors, namely readthrough
of stop codons.

Highlights
d Stop codon readthrough is heterogeneous among single cells

d Increased UGA readthrough promotes bacterial growth

d Reducing protein synthesis levels enhances UGA

readthrough

d Fluctuations of translational components lead to UGA

readthrough heterogeneity

Fan et al., 2017, Molecular Cell 67, 826836

September 7, 2017 2017 Elsevier Inc.
http://dx.doi.org/10.1016/j.molcel.2017.07.010

Heterogeneity of Stop Codon Readthrough
in Single Bacterial Cells
and Implications for Population Fitness
Yongqiang Fan,1,7 Christopher R. Evans,1,2,7 Karl W. Barber,3,4,7 Kinshuk Banerjee,5 Kalyn J. Weiss,1,2 William Margolin,1,2
Oleg A. Igoshin,5,6 Jesse Rinehart,3,4 and Jiqiang Ling1,2,8,*
1Department of Microbiology and Molecular Genetics, McGovern Medical School, University of Texas Health Science Center, Houston,
TX 77030, USA
2Graduate School of Biomedical Sciences, Houston, TX 77030, USA
3Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06520, USA
4Systems Biology Institute, Yale University, West Haven, CT 06516, USA
5Center for Theoretical Biological Physics, Rice University, Houston, TX 77005, USA
6Department of Bioengineering, Rice University, Houston, TX 77005, USA
7These authors contributed equally
8Lead Contact
*Correspondence: jiqiang.ling@uth.tmc.edu
http://dx.doi.org/10.1016/j.molcel.2017.07.010
SUMMARY
Gene expression noise (heterogeneity) leads to
phenotypic diversity among isogenic individual cells.
Our current understanding of gene expression noise
is mostly limited to transcription, as separating trans-
lational noise from transcriptional noise has been
challenging. It also remains unclear how translational
heterogeneity originates. Using a transcription-
normalized reporter system, we discovered that
stop codon readthrough is heterogeneous among
single cells, and individual cells with higher UGA
readthrough grow faster from stationary phase. Our
work also revealed that individual cells with lower
protein synthesis levels exhibited higher UGA
readthrough, which was confirmed with ribosome-
targeting antibiotics (e.g., chloramphenicol). Further
experiments and mathematical modeling suggest
that varied competition between ternary complexes
and release factors perturbs the UGA readthrough
level. Our results indicate that fluctuations in the con-
centrations of translational components lead to UGA
readthrough heterogeneity among single cells, which
enhances phenotypic diversity of the genetically
identical population and facilitates its adaptation to
changing environments.
INTRODUCTION
Protein synthesis is a fundamental and essential process in all
three domains of life. Accurate protein synthesis requires correct
matching of amino acids and transfer RNAs (tRNAs) by amino-
acyl-tRNA synthetases, proofreading of aminoacyl-tRNAs (aa-
tRNAs) by trans-editing factors, precise decoding of mRNA
826 Molecular Cell 67, 826836, September 7, 2017 2017 Elsevier Inc.
Molecular Cell
Article
codons by proper aa-tRNAs, and accurate translocation of
mRNA by the ribosome (Fredrick and Noller, 2002; Ling et al.,
2009; Liu et al., 2015; Mascarenhas et al., 2008; Zaher and
Green, 2009). High translational errors cause growth defects
and cell death in bacteria (Bacher et al., 2005; Davis, 1987; Kar-
khanis et al., 2007), mitochondrial dysfunction in yeast (Reynolds
et al., 2010), shortened lifespan in flies (Lu et al., 2014), and neu-
rodegeneration and cardioproteinopathy in mammals (Lee et al.,
2006; Liu et al., 2014). Surprisingly, naturally isolated Escherichia
coli strains display a wide range of ribosomal fidelity, suggesting
that high translational errors may be favored under some natural
habitats (Mikkola and Kurland, 1992). Recent evidence suggests
that increased translational errors paradoxically provides bene-
fits to microorganisms under certain stress conditions (Bezerra
et al., 2013; Ling et al., 2015; Pan, 2013; Ribas de Pouplana
et al., 2014). For example, amino acid misincorporation in the
b subunit of RNA polymerase increases resistance of mycobac-
teria to rifampicin (Javid et al., 2014; Su et al., 2016), and trans-
lational errors improve bacterial tolerance to oxidative stress by
activating the general stress response (Fan et al., 2015; Fredriks-
son et al., 2007). Interestingly, in such cases only subpopulations
of genetically identical cells survive severe stresses (Fan et al.,
2015; Su et al., 2016), suggesting that stress response activated
by translational errors may be heterogeneous (noisy) in individual
cells. However, the noise levels of translational errors have not
been determined, and how such heterogeneity originates re-
mains unknown.
Gene expression has been shown to be stochastic and noisy
(Balazsi et al., 2011; Kaern et al., 2005; Levine and Hwa, 2007;
Levine et al., 2013; Newman et al., 2006; Sanchez and Golding,
2013; Taniguchi et al., 2010; Young et al., 2013). The first exper-
imental evidence came from pioneering work by Elowitz et al.
(2002) showing that transcription is intrinsically noisy. Later
studies revealed that transcription is bursty and noncontinuous
(Blake et al., 2003; Levine et al., 2013; Sanchez and Golding,
2013), and the promoter architecture regulates transcriptional
noise (Sanchez et al., 2013). Noise in gene expression can be

harmful by disrupting regulatory networks, but increasing evi-
dence shows that such noise can also be beneficial by gener-
ating phenotypic heterogeneity that helps the population to
quickly adapt to environmental changes through bet-hedging
(Ackermann, 2015; Blake et al., 2006; Sanchez and Golding,
2013). Compared with transcriptional noise, translational noise
is extremely poorly understood. Earlier studies used a single
fluorescent reporter to determine the noise levels when transla-
tion initiation (Ozbudak et al., 2002) or the codon context (Blake
et al., 2003) is varied. It was suggested that in Bacillus subtilis,
increasing the efficiency of translation initiation enhances trans-
lational noise (Ozbudak et al., 2002). However, it remains a
significant challenge to separate translational noise from tran-
scriptional noise (Paulsson, 2004). Here, we have developed
transcription-normalized dual-fluorescence reporters to quanti-
tate the noise levels of stop codon readthrough and investigate
the sources of such noise. Our work suggests that reduced
translation promotes UGA readthrough by altering competition
between the ternary complex (TC) and release factors and pro-
vides a model for the heterogeneity of UGA readthrough among
single cells. We also show that increased UGA readthrough pro-
Figure 1. Dual-Fluorescent Reporters for
Translational Errors
(A) Dual-fluorescence reporters that measure
translational errors. MCherry (m) and yfp (y) genes
are fused under the control of a constitutive pro-
moter PLtetO-1. At the end of mCherry is a stop
codon, a frameshifting (fs) codon, or no stop
codon. Readthrough of the stop codon or frame-
shifting produces a single mCherry-YFP fusion
protein and yields YFP signal.
(B) The reporters on a low-copy-number plasmid
were expressed for 24 hr in Luria-Bertani broth
(LB) at 37! C in wild-type (MG1655) and ribosomal
error-prone (rpsD*) E. coli strains. Fluorescence
was quantified by spectrometry on a plate reader.
The error rate was calculated as the YFP/mCherry
ratio of the error reporter normalized by the YFP/
mCherry ratio of the m-y control.
(C) Western blotting showing that UGA read-
through of the m-TGA-y reporter. Anti-mCherry
antibody was used to detect both mCherry-YFP
fusion and mCherry. Data are represented as
mean SD.
(D and E) Label-free quantitative mass spectrom-
etry analysis of UGA readthrough in the dual-
fluorescence reporter protein m-TGA-y (D) and
native protein RpsG (E). Extracted ion chromato-
grams (XIC) for the m-TGA-y reporter peptide
WLQTSAGEAAAK (W, or tryptophan, marks the
UGA readthrough site in the peptide) and the
RpsG peptide QPALGYLNWTPK are shown from
two different strains. XIC peak area indicates the
relative abundance of the detected peptide.
Relative peak intensities were fixed at 100% indi-
vidually for comparative purposes.
motes growth of E. coli cells from stationary phase, indicating
that UGA readthrough heterogeneity provides an advantage to
the population during environmental shifts.
RESULTS
Dual-Fluorescence Reporters to Quantitate
Translational Errors
To separate the noise of translational errors from transcriptional
noise, we have constructed a series of mCherry-YFP (red and
yellow fluorescent proteins) fusion reporters that measure the
errors of stop-codon readthrough and frameshifting (Figure 1).
The mCherry and yfp genes are transcribed as a single mRNA
and translated from the same start codon to yield a single poly-
peptide (Figure 1A), therefore minimizing the influence of the
noise from transcription and translation initiation in single-cell
analysis. Additionally, both mCherry and YFP are stable in
E. coli (Figures S1A and S1B), and degradation of the reporter
proteins should produce a minimal level of noise. Expression of
the dual-fluorescence reporter on a low-copy-number plasmid
does not affect bacterial growth (Figure S1C). With these
Molecular Cell 67, 826836, September 7, 2017 827

Figure 2. UGA Readthrough Is Heterogeneous among Single Cells
(A and B) YFP and mCherry fluorescence in MG1655 cells carrying either the m-y (A) or m-TGA-y (B) reporter. Cells were grown in LB for 24 hr. The YFP/mCherry
ratio is the relative YFP signal normalized by the mCherry signal. Cells with high and low UGA readthrough are indicated by yellow and red arrows, respectively.
(C and D) In the scatterplots of m-y (C) and m-TGA-y (D), each dot represents a single cell. The fluorescence is background-subtracted and arbitrary units
are shown.
(E and F) Heterogeneity of UGA readthrough among single cells is indicated by CV (E) and noise (F) of the YFP/mCherry ratio. m, the mean of the YFP/mCherry
ratio; s, SD. Data are represented as mean SD. AU, arbitrary units.
reporters, 0.2%3% error rates were detected in wild-type (WT)
E. coli MG1655 grown in Luria-Bertani broth (LB) at 37! C using
fluorescence spectrometry (Figures 1B and S1D). Such error
rates were orders of magnitude higher than DNA mutational
and transcriptional error rates (Gordon et al., 2015; Rosenberg
et al., 2012), suggesting that the observed errors directly result
from translation. In support of this notion, a ribosomal ambiguity
mutation (rpsD I199N or rpsD*), which affects the ribosomal de-
coding center to reduce translational fidelity (Bjorkman et al.,
1999; Fan et al., 2015; Kramer and Farabaugh, 2007), increased
all four types of errors that we tested (Figure 1B).
To validate that the fluorescence of the reporters reflects the
actual protein level, we used western blotting to detect and
quantitate mCherry and mCherry-YFP fusion protein (Figure 1C).
In WT cells expressing the m-TGA-y reporter, the mCherry-YFP
fusion accounted for 2.5% of total mCherry proteins produced.
This was in good agreement with the 2% UGA readthrough level
determined by fluorescence spectrometry (Figure 1B) and
consistent with previous reports (Devaraj et al., 2009; Eggerts-
son and Soll, 1988). The fraction of mCherry-YFP in the rpsD*
strain increased to 8%, which was again consistent with the
UGA readthrough level determined by fluorescence (Figure 1B).
To identify the amino acid(s) incorporated at UGA sites as well
as to detect UGA readthrough events in the native proteome, we
further applied quantitative mass spectrometry of WT and rpsD*
cells with and without the m-TGA-y reporter (Figures 1D, 1E, and
S2; Table S1). UGA readthrough in the m-TGA-y reporter and
native proteins were indeed detected with liquid chromatog-
raphy-tandem mass spectrometry (LC-MS/MS). Our unbiased
MS/MS search shows that tryptophan (W) is the predominant
amino acid that suppresses UGA codons in E. coli. The level of
the peptide resulting from UGA readthrough in the m-TGA-y re-
828 Molecular Cell 67, 826836, September 7, 2017
interesting to note that for 2D, you see that, even though the units are arbitrary, assuming they are
comparable, there is a lot more M than Y. also note that we could still have R2 value close to one
even in this condition, but a low R value tells us that the read-through levels VARY between individual
cell, which is why the relative level of red and yellow change from cell to cell.
porter was increased 3-fold in the rpsD* strain compared to the
WT (Figure 1D), in line with the fluorescence and western blotting
results (Figures 1B and 1C). These results demonstrate that our
dual-fluorescence reporter system is robust for quantitation of
translational errors.
UGA Readthrough Is Heterogeneous among Single Cells
We next applied our dual-fluorescence reporters to determine
the heterogeneity of stop codon readthrough among genetically
identical single cells grown under the same condition. Using fluo-
rescence microscopy, we visualized mCherry and YFP signals in
single cells (Figures 2 and S1E). Whereas the YFP signal of the
m-TGA-y reporter was substantially higher than background,
cells without reporters showed no detectable fluorescence
signal (Figure S1E), suggesting that the influence of auto fluores-
cence on signal quantification is negligible under our experi-
mental setting. Our results showed that the control m-y reporter
exhibited tight linear correlation between the YFP and mCherry
signals, and the YFP/mCherry ratio was mostly homogeneous
(Figures 2A, 2C, and S3A). In contrast, the YFP/mCherry ratio
of the m-TGA-y reporter (indicating UGA readthrough level)
was more dispersed (Figures 2B, 2D, and S3B), suggesting
that the concentration of UGA readthrough products was hetero-
geneous among single cells.
Heterogeneity of gene expression is quantified with coefficient
of variation (CV), calculated as the ratio of the SDn (s) over the
mean (m) or noise (s2/m2) (Blake et al., 2003; Elowitz et al.,
2002; Taniguchi et al., 2010). Our results revealed that the CV
of YFP/mCherry ratio in cells with the control m-y reporter was
"0.1 (Figure 2E). Such a low level of heterogeneity may be
caused by noise from fluorophore maturation, partial degrada-
tion of mRNA and protein, ribosomal drop-off during translation,
missense errors, and the imaging system. The CV of YFP/
mCherry ratio in cells with the m-TGA-y reporter increased to
0.2, corresponding to a 4-fold increase in noise (Figures 2E
and 2F). It has been suggested that when the copy number of
protein molecules is low (<1,000/cell), partitioning errors and
finite-number effect would contribute to the overall protein noise,
whereby reducing the fluorescence mean would increase the CV
and noise (Huh and Paulsson, 2011; Kaern et al., 2005). How-
ever, the estimated concentration of mCherry-YFP fusion protein
in cells with the m-TGA-y reporter is "6,000 molecules per cell
(see the STAR Methods). Therefore, partitioning noise and
finite-number effect is expected to have little contribution to
the observed CV of the YFP/mCherry ratio. In support of this
argument, we found that reducing the mean of mCherry by
decreasing the promoter strength of the m-TGA-y reporter did
not further increase the CV (Figure S3C). We thus reason that
the higher CV and noise of the YFP/mCherry ratio in m-TGA-y
compared to m-y is largely due to the heterogeneity of UGA
readthrough events among individual cells.
Reduced Protein Synthesis Increases UGA Readthrough
To understand how the heterogeneity of UGA readthrough
arises, we analyzed the correlation between UGA readthrough
(indicated by the YFP/mCherry ratio) and various cell parameters
(Figures 3 and S3). We found that cells with lower mCherry
expression levels exhibited higher UGA readthrough (Figures
3A and 3B). To test whether high UGA readthrough reduces
mCherry expression or reduced protein expression increases
UGA readthrough, we first used a UGA suppressor tRNASer
variant with a UCA anticodon (Figures S3G and S3H). The sup-
pressor tRNA substantially increased UGA readthrough, but
did not affect the mCherry protein level. We next tested whether
reducing protein expression would enhance UGA readthrough.
A low mCherry level may result from reduced transcription
or translation. We demonstrated that reducing transcription
with rifampicin (Rif), which inhibits RNA polymerase, did not
significantly affect UGA readthrough (Figure S3I). In contrast,
treating E. coli with low concentrations of chloramphenicol
(Chl), which binds to the peptidyl transferase center (PTC) of
the ribosome and impedes binding of aa-tRNAs and release fac-
tors (Wilson, 2014), reduced protein synthesis and increased
UGA readthrough, as determined by fluorescence spectrometry
and western blotting (Figures 3C and 3D). To verify that the
observed increase in UGA readthrough by Chl was not an artifact
due to the sequence context of the dual-fluorescence reporter,
we tested UGA readthrough at a native chromosomal site of
the cspC gene (Figure 3E) and confirmed that Chl also signifi-
cantly enhanced readthrough of the native UGA codon (Figures
3E and 3F). To determine whether increased UGA readthrough
is a specific effect of Chl, we further tested other ribosomal
inhibitors tetracycline (Tet, inhibiting aa-tRNA delivery to the
ribosome), spectinomycin (Spc, inhibiting translocation), and
erythromycin (Ery, blocking the 50S exit tunnel). Tet, Spc,
and Ery also significantly enhanced UGA readthrough as Chl
(Figure S4A).
The efficiency of stop codon readthrough is a direct result of
competition between TC (composed of elongation factor Tu
[EF-Tu], aa-tRNA, and GTP) and release factors. UGA stop
codons are suppressed by Trp-tRNATrp (Figures 1D, 1E, and
S2; Table S1) and terminated by release factor 2 (RF2) (Engel-
berg-Kulka, 1981; Youngman et al., 2007). It has been reported
that in E. coli K-12 strains (including MG1655), RF2 contains
an A246T mutation that decreases termination efficiency
(Dincbas-Renqvist et al., 2000; Mora et al., 2007). To test
whether reduced protein synthesis specifically increases UGA
readthrough in K-12 strains, we examined E. coli BL21 and
Salmonella enterica Serovar Typhimurium LT2 strains that both
carry wild-type RF2. Like MG1655, both BL21 and S. Typhimu-
rium strains exhibited strong negative correlation between
UGA readthrough and mCherry intensity and increased UGA
readthrough upon Chl treatment (Figure S5), demonstrating
that reduced protein synthesis enhances UGA readthrough inde-
pendent of the A246T mutation.
The next question is how reduced translation may increase
UGA readthrough errors, which is surprising given previous ki-
netic studies showing that there is a trade-off between transla-
tional speed and accuracy (Johansson et al., 2008). Using acidic
gel northern blotting, we show that the overall Trp-tRNATrp level
increases in the presence of Chl due to an increase in total
tRNATrp (Figure 4A). Further time course analysis shows that
tRNATrp is stable both in the presence and absence of Chl (Fig-
ure 4B), indicating that the increased level of tRNATrp with Chl is
not caused by increased tRNA stability, but rather by enhanced
transcription. We next performed mathematical modeling to un-
derstand how reduced translation may increase UGA read-
through (Figures S4B and S4C; Table S2). Our modeling suggests
that increasing TC concentration enhances UGA readthrough
rate. Based on these experimental and modeling results, we
reason that reduced translation increases the effective concen-
tration of EF-Tu:Trp-tRNATrp:GTP complex to more efficiently
compete against RF2, thereby promoting UGA readthrough.
A recent study shows that Chl and Tet decrease the active
fraction of ribosomes (Dai et al., 2016). To test the effects of
active ribosomes on UGA readthrough, we used mutant strains
lacking several copies of the rrn operon that encodes ribosomal
RNAs (Quan et al., 2015). Deleting six out of seven copies of the
rrn operon significantly increased UGA readthrough (Figure 3G),
suggesting that reducing the cellular ribosome concentration
promotes UGA readthrough. We also tested a strain that
lacks RMF (ribosome modulation factor), which promotes 70S
ribosomes to form inactive dimers during stationary phase
and stress conditions (Polikanov et al., 2012; Yoshida and
Wada, 2014). Deleting rmf is expected to increase the concen-
trations of active ribosomes. Indeed, deleting rmf mitigates the
effect of Chl to enhance UGA readthrough (Figure 3H), support-
ing that Chl increases UGA readthrough by reducing the fraction
of active ribosomes. It has been suggested that low codon adap-
tation of highly transcribed genes (e.g., our m-TGA-y reporter,
expressed at over 300,000 protein molecules per cell) may also
sequester ribosomes and reduce the active fraction (Kudla
et al., 2009). In contrast, reduced translation initiation of the re-
porter is not expected to affect the active ribosome concentra-
tion. We therefore reduced expression of the reporter proteins
by either introducing non-optimal codons to mCherry or
decreasing translation initiation efficiency. Non-optimal codons
enhanced UGA readthrough as Chl (Figure S4D), but reducing
Molecular Cell 67, 826836, September 7, 2017 829
 Just see a) and b). this is an example of finite number effect, as you can see if the protein
concentration is high, there is very low fluctuation in the total abundance but if the protein
concentration is low, then the fluctuations start becoming big. side note - the abundance values
are the same between a and b, because in b) aside from decreasing the transcription rate by 100,
they also decreased the cell volume by 100, to keep the abundance the same, so that we can
compare similar values in a) and b) source - https://www.nature.com/nrg/journal/v6/n6/fig_tab/
nrg1615_F2.html












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 they did the E and F to check if the
increased UGA read-through was
because of something specific about the
sequence they were using. So they tried
to check UGA read-through in another
sequence which is always transcribed -
cspC, and still saw UGA read-through

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Figure 3. Reducing Protein Synthesis Increases the Level of UGA Readthrough

(A) In single MG1655 cells, higher UGA readthrough correlates (Spearmans rank correlation) with lower mCherry protein level. The mCherry intensity is the
mCherry signal normalized by cell volume with arbitrary units. Relative UGA readthrough is calculated from the YFP/mCherry ratio. Experimental conditions were
the same as in Figure 2.
(B) The y axis (R10%) is the ratio of the average UGA readthrough in the bottom 10% of cells divided by the average UGA readthrough in the top 10% of cells ranked
by mCherry intensity from (A).
(C) MG1655 cells were grown in LB with and without Chl for 24 hr, and UGA readthrough and protein synthesis rates were determined by fluorescence spec-
trometry. Treating MG1655 with low doses of Chl decreases protein synthesis rate and enhances UGA readthrough.
(D) Western blotting confirms that Chl increases UGA readthrough of the m-TGA-y reporter.
(E and F) UGA readthrough of chromosomally encoded CspC increases in the presence of chloramphenicol.
(E) The nucleotide sequence of the 30 end of the chromosomal constitutively expressed cspC gene is shown. Red letters indicate the first and second stop
codons. An in-frame YFP tag is inserted immediately after the first TGA codon of the native cspC gene (CspC-TGA-YFP-1) or before the second stop codon
(CspC-TGA-YFP-2) in MG1655. Western blotting result shows that readthrough of the cspC UGA codon is significantly increased by addition of Chl.
(F) Quantitation of western blotting results in (E).
(G) Reducing ribosome copy number increases UGA readthrough. WT MG1655 and its ribosomal operon deletion mutants (D2, DrrnEG; D4, DrrnGBAD; D6,
DrrnGADBHC) were grown in LB with and without Chl for 24 hr, and UGA readthrough levels were determined with fluorescence spectrometry.
(H) Deleting rmf partially suppresses the effect of Chl to increase UGA readthrough.
(I) Chl treatment decreases the CV of UGA readthrough. Data are represented as mean SD. AU, arbitrary units.

translation initiation efficiency by changing the Shine-Dalgarno Next, we investigated how reduced translation affects the
sequence did not alter the UGA readthrough level (Figure S4E). heterogeneity of UGA readthrough. Both Chl and codon non-
These results are consistent with the notion that reducing the optimization decreases the CV of YFP/mCherry ratio (Figures
concentration of active ribosomes promotes UGA readthrough. 3I and S4F), suggesting that UGA readthrough heterogeneity is

830 Molecular Cell 67, 826836, September 7, 2017


reduced when translation is attenuated across the population.
Collectively, these results suggest that reduced translation en-
hances the level of UGA readthrough, and various levels of pro-
tein synthesis among single cells contribute to heterogeneous
UGA readthrough in a bacterial population.
Defective Translation Termination Increases UGA
Readthrough Heterogeneity
We next tested how RF2 fluctuations affect UGA readthrough. In
E. coli, the coding region of the prfB gene (encoding RF2) con-
tains a TGAC frameshifting codon, which autoregulates the pro-
duction of RF2 (Curran, 1993; Curran and Yarus, 1988). A high
level of RF2 decreases the frameshifting efficiency at the
TGAC site by promoting early termination, therefore reducing
RF2 translation. To test how RF2 autoregulates UGA read-
through heterogeneity, we used a genome engineering tool
(Wang et al., 2009) to change the chromosomal site of TGAC in
prfB to TAGC. RF2 does not terminate translation at UAG co-
dons. Therefore, the TAGC mutation in prfB is expected to
abolish autoregulation of RF2 production and increase fluctua-
tions of RF2 protein levels among single cells. Surprisingly, the
TAGC mutation did not significantly alter the level or CV of
UGA readthrough (Figure S6), suggesting that the RF2 activity
was possibly close to saturation for translation termination at
UGA codons in the WT cells. In line with this, overexpressing
RF2 in WT cells did not further decrease the level of UGA read-
through (Figures 5A and 5B). However, when prfC (encoding
RF3 that facilitates RF2 during termination) was deleted, overex-
pressing RF2 decreased the level of UGA readthrough, suggest-
Figure 4. Chloramphenicol Increases Trp-
tRNATrp Level
(A) Chl treatment increases the level of Trp-
tRNATrp shown by acidic northern blotting. Total
RNA was isolated from MG1655 cells grown in LB
with or without 2 mg/mL Chl under acidic condi-
tions andtreated with or without alkaline (OH#)
before acid gel electrophoresis and northern
blotting. Alkaline treatment causes deacylation of
aminoacyl-tRNAs. Almost 100% of tRNATrp was
aminoacylated without alkaline treatment. SsrA
was used as an internal standard to calculate the
relative concentration of Trp-tRNATrp.
(B) Stability of tRNATrp with and without Chl.
MG1655 cells grown in LB in the presence
absence of Chl were treated with a high concen-
tration of rifampicin to stop transcription. RNA
samples were prepared at indicated time points
following addition of Rif and subjected to northern
blotting analysis. Chl increases the overall level of
tRNATrp, but not the stability.
(C) The level of Gly-tRNAGly was determined using
acidic gel northern blotting as in Figure 4.
(D) The protein levels of EF-Tu and FLAG-RF2 with
and without Chl revealed by western blotting.
A FLAG tag is fused to the C terminus of RF2 at the
native chromosomal site. Data are represented as
mean SD.
ing that the RF2 activity was no longer saturating in the absence
of RF3. Consequently, deleting prfC increased both the level and
heterogeneity of UGA readthrough (Figures 5C and 5D). These
results suggest that RF2 fluctuations do not significantly
contribute to UGA readthrough heterogeneity in WT E. coli cells,
but defects in translation termination enhance the sensitivity of
UGA readthrough to RF2 fluctuations among single cells,
thereby increasing readthrough heterogeneity.
UGA Readthrough Promotes Cell Growth from
Stationary Phase
Altering translational fidelity results in various phenotypic
changes (Drummond and Wilke, 2009; Pan, 2013). To provide in-
sights into how UGA readthrough heterogeneity affects bacterial
physiology, we used time-lapse fluorescence microscopy to
monitor division of individual cells with various levels of UGA
readthrough. Stationary-phase WT cells carrying the m-TGA-y
reporter were tested on agar pad with minimal glucose medium
in the chamber of an automated fluorescence microscope. We
found that cells with high levels of UGA readthrough required a
shorter time to reach the first division than cells with low UGA
readthrough levels (Figure 6A). To test whether increased UGA
readthrough is sufficient to cause faster growth from stationary
phase, we expressed the WT and suppressor tRNASer in
MG1655. The suppressor tRNA indeed improved regrowth of
stationary-phase cells in minimal media with various carbon
source (Figures 6B, 6C, and S7A). However, growth in rich media
LB was not affected by the suppressor tRNA (Figure S7A),
suggesting that the growth advantage provided by UGA
Molecular Cell 67, 826836, September 7, 2017 831

RF3 also maintains fidelity during translation elongation (Zaher and Green, 2011). The rpsD* result suggests that increased UGA readthrough heterogeneity upon
RF3 deletion is caused by defective termination rather than reduced fidelity during elongation. Data are represented as mean SD.
readthrough is manifested under poor-nutrient conditions. In
addition, regrowth of log-phase cells was not much affected
by the suppressor tRNA even in minimal media (Figure S7B).
Collectively, these results suggest that heterogeneous UGA
readthrough allows a subpopulation of cells to grow faster
from the stationary phase, thereby improving the overall fitness
of the bacterial population.
DISCUSSION
Noise in gene expression has been extensively studied at the
transcriptional level, but the levels and sources of noise during
translation are poorly understood. Here, we have developed a
dual-fluorescence reporter system to determine the noise of
stop codon readthrough with minimal influence from noise pro-
duced during transcription, translation initiation, and protein
degradation. Such reporters will be broadly useful to determine
the levels and heterogeneity of translational errors in single cells
in their native environments, e.g., within biofilms and during host-
microbe interactions. We have further provided in-depth ana-
lyses of the regulation of UGA readthrough noise. The sources
of gene expression noise include intrinsic sources that result
from stochastic transcription and translation of individual genes
and extrinsic sources caused by fluctuations of global resources
among single cells (Ackermann, 2015; Blake et al., 2003; Elowitz
et al., 2002). The heterogeneity of UGA readthrough over long
periods of growth under our experimental conditions is likely
dominated by extrinsic sources of noise, such as fluctuations
of the concentrations of nutrients and translational components
among individual cells. For example, amino acid levels may
reduce or deplete in some cells after 24 hr of growth in LB.
UGA readthrough results from occasional recognition of the
832 Molecular Cell 67, 826836, September 7, 2017
Figure 5. Defective Termination Increases
UGA Readthrough Heterogeneity
(A) ASKA-prfB (Kitagawa et al., 2005) leads to
overproduction of prfB mRNA as determined with
quantitative reverse transcriptase PCR. In the
absence of isopropyl b-D-1-thiogalactopyrano-
side (IPTG), the promoter of the ASKA plasmid is
leaky (Kitagawa et al., 2005).
(B) The m-TGA-y reporter was tested in strains
with and without the RF2 overexpression plasmid.
In MG1655, increasing the RF2 level does not
further decrease UGA readthrough, suggesting
that the RF2 activity is close to saturation. In
contrast, excess levels of RF2 decrease UGA
readthrough in the RF3 (encoded by prfC) deleting
strain, indicating increased sensitivity of UGA
readthrough to RF2 fluctuations when the release
factor activity is compromised. The DprfC result
also suggests the RF2 protein is successfully
overproduced from the plasmid.
(C) Deleting RF3 and introducing the rpsD* muta-
tion both increases UGA readthrough of the
m-TGA-y reporter.
(D) RF3 deletion increases the CV of UGA read-
through. In contrast, the rpsD* mutation decreases
the CV. In addition to recycling release factors,
stop codon by the EF-Tu:Trp-tRNATrp:GTP ternary complex,
which competes with RF2 that releases the elongating peptide
(Figure 7). Fluctuations of TC and RF2 among single cells would
lead to heterogeneity of UGA readthrough and increase its noise.
We have shown that reduced translation increases the level of
UGA readthrough and Trp-tRNATrp (Figures 3, 4, S4, and S5).
Because tRNATrp is stable (Figure 4B), the increase in tRNATrp
in the presence of Chl is likely due to enhanced transcription.
In addition to tRNATrp, the level of tRNAGly is also increased by
Chl (Figure 4C). It is possible that some unknown protein factor
is involved in repressing the tRNA promoters, and Chl reduces
synthesis of the repressor to upregulate tRNA transcription.
We also show that the protein level of EF-Tu is not significantly
affected by Chl (Figure 4D). Given that EF-Tu is very abundant
and in 6-fold excess relative to the ribosome (Dai et al., 2016),
an increase in Trp-tRNATrp is expected to drive the formation
of TC. Another factor that may affect UGA readthrough is the
RF2 protein level. We show that the FLAG-tagged RF2 level in-
creases in the presence of Chl (Figure 4D), which is not expected
to enhance UGA readthrough. Our data thus suggest that
reduced protein synthesis promotes UGA readthrough by
increasing the effective concentration of TC.
Recently, Dai et al. (2016) have shown that some ribosome-
targeting antibiotics, including Chl and Tet, reduce the active
fraction of ribosomes rather than decreasing translation elonga-
tion rates). Our results suggest that reducing the concentration
of active ribosomes promotes UGA readthrough (Figures 3, 4,
and S5). Several lines of evidence indicate that protein synthesis
is intrinsically heterogeneous: (1) the mCherry protein level is
highly heterogeneous among single cells even with the chromo-
somal reporters (Figure S3A), indicating that overall protein pro-
duction is heterogeneous; (2) the components of the protein

synthesis machinery, including ribosomal proteins and elonga-
tion factors, vary among single cells at the protein level in
E. coli (Taniguchi et al., 2010); (3) translation initiation has been
shown to be noisy in bacteria (Ozbudak et al., 2002); and (4) ribo-
some hibernation alters gene expression heterogeneity (Guido
et al., 2007). Collectively, such evidence suggests that the
concentration of active ribosomes may vary among genetically
Figure 6. UGA Readthrough Promotes Cell
Growth from Stationary Phase
(A) Percentage of cells with high and low error
levels that require different time periods to reach
the first cell division. Stationary-phase MG1655
cells were placed on M9 glucose agar pad and
monitored for fluorescence and growth. High and
low error cells are the top and bottom quartiles
of individual cells ranked by the YFP/mCherry
ratio of the m-TGA-y reporter, respectively.
A significantly higher percentage of cells with
high UGA readthrough levels divide within
120 min compared with cells with low UGA
readthrough levels.
(B) Stationary-phase cultures of MG1655 carrying
the WT or UGA suppressor tRNASer were diluted
50-fold, and growth was monitored over time.
(C) UGA suppressor tRNASer improves growth in
minimal media with various carbon sources
(1% each), but not in LB. Data are represented as
mean SD.
identical single cells due to heterogeneity during expression of
ribosomal proteins and rRNAs, ribosome maturation, and inacti-
vation. This would exemplify fluctuations of TC and contribute to
the heterogeneity of stop codon readthrough (Figure 7).
Our proteomic analysis identified Trp to be the major amino acid
that suppresses UGA in E. coli, which is in agreement with previous
studies (Engelberg-Kulka, 1981). E. coli tRNATrp contains a
Figure 7. Model for UGA Readthrough Het-
erogeneity
(A) The near-cognate Trp-tRNATrp forms a ternary
complex (TC) with EF-Tu and GTP to compete with
RF2 for the UGA stop codon. Recognition of UGA
by RF2 leads to release of the growing peptide
from the ribosome, whereas Trp-tRNATrp sup-
presses UGA by adding Trp to the growing pep-
tide. The UGA readthrough level in a single cell is
determined by the effective concentrations of TC
and RF2.
(B) The concentration of active ribosomes may
vary among single cells due to fluctuations of
global resources and ribosome maturation/inacti-
vation. A low concentration of active ribosomes
decreases global protein synthesis, which in-
creases the effective concentration of TC due to its
reduced usage during translation.
(C) UGA readthrough is sensitive to TC fluctua-
tions. An increase in effective TC leads to
enhanced UGA readthrough.
(D) WT cells with effective translation termination
exhibit nearly saturated RF2 activity, and UGA
readthrough is insensitive to RF2 fluctuations.
Defects in RF2 activity, e.g., due to RF3 deletion,
enhance the sensitivity of UGA readthrough to RF2
fluctuations and thus increase UGA readthrough
heterogeneity. Dashed lines indicate arbitrary
ranges of concentrations. AU, arbitrary units.
Molecular Cell 67, 826836, September 7, 2017 833
modification at A37 that facilitates C-A pairing at the wobble posi-
tion (Vacher et al., 1984), which may explain why tRNATrp sup-
presses UGA better than other near-cognate tRNAs such as
tRNACys. The suppression efficiency of UAG appears to be lower
than UGA, presumably because of the stronger termination
activity of RF1. Indeed, in an RF1 deletion strain, multiple amino
acids have been detected to suppress UAG (Aerni et al., 2015).
In E. coli, UGA is used by "30% of the protein-coding genes
as the stop codon. Readthrough of stop codons may cause mis-
folded protein stress or production of protein isoforms with new
functions (Dunn et al., 2013; True and Lindquist, 2000). For
example, sequences following stop codon readthrough may
contain novel localization signals (Dunn et al., 2013). In this
study, we show that increased UGA readthrough in E. coli en-
hances growth under poor-nutrient conditions and promotes
growth from stationary phase (Figures 6B, 6C, and S7A). The un-
derlying mechanism remains to be elucidated. A pathway enrich-
ment analysis suggests that genes with UGA as stop codons are
most significantly enriched in ABC transporters (Table S3), which
are involved in nutrient uptake (Hollenstein et al., 2007; Jones
and George, 2004). It is possible that UGA readthrough may alter
the function of such transporters and provide growth advantage
when nutrients are limited. In line with this notion, UGA suppres-
sion does not enhance growth in rich media (Figure S7A). Such
advantage of high UGA readthrough on cell growth is also
observed in single cells (Figure 6A). Heterogeneous UGA read-
through among single cells may thus provide benefits to the
microbial population by enhancing phenotypic diversity and
facilitating adaptation to ever-changing environments.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Bacterial Strains, Plasmids and Growth Conditions
d METHOD DETAILS
B Fluorescence Microscopy
B Time-lapse Microscopy
B Rate Determination
B Calculation of CV and Noise
B Detection of Native UGA Readthrough in cspC
B RNA Isolation
B qRT-PCR
B Acidic Gel Northern Blotting
B Protein Digestion and Proteomics
B Peptide Database Construction and Searches
B Mathematical Modeling
d QUANTITATION AND STATISTICAL ANALYSES
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and four tables and can be
found with this article online at http://dx.doi.org/10.1016/j.molcel.2017.
07.010.
834 Molecular Cell 67, 826836, September 7, 2017
AUTHOR CONTRIBUTIONS
Y.F., C.R.E., K.W.B., W.M., O.A.I., J.R., and J.L. designed the experiments.
Y.F., C.R.E., K.W.B., K.J.W., and J.L. performed the experiments. Y.F.,
C.R.E., K.W.B., K.J.W., J.R., and J.L. analyzed the experimental data. K.B.
and O.A.I. performed mathematical modeling. Y.F., C.R.E., K.W.B., K.B.,
W.M., O.A.I., J.R., and J.L. wrote the manuscript.
ACKNOWLEDGMENTS
We thank Drs. Arvind R. Subramaniam and Philippe Cluzel (Harvard University)
and the National BioResource Project (Japan) for plasmids, Dr. Nicholas De
Lay (The University of Texas Health Science Center at Houston) for strains,
Dr. Veronica Rowlett (The University of Texas Health Science Center at Hous-
ton) for technical assistance, and Dr. Marc Lajoie (University of Washington) for
help with bioinformatics. This work was funded by NIGMS (R01GM115431 to
J.L., R01GM61074 to W.M., and R01GM117230 to J.R.) and NSF (DGE-
1122492 to K.W.B. and PHY-1427654 to O.A.I).
Received: March 20, 2017
Revised: May 22, 2017
Accepted: July 7, 2017
Published: August 3, 2017
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Mouse monoclonal anti-mCherry (1C51) NOVUS Biologicals Cat# NBP196752; RRID: AB_11034849
Mouse monoclonal anti-GFP Roche Cat# 11814460001; RRID: AB_390913
Mouse monoclonal anti-RpoB Santa Cruz Biotechnology Cat# SC-101613; RRID: AB_1129055
Mouse monoclonal anti-FLAG Sigma Cat# A8592; RRID: AB_439702
Mouse monoclonal anti-EF-Tu Hycult Biotech Cat# HM6010; RRID: AB_1953571
Bacterial and Virus Strains
rpsD* (Fan et al., 2015) N/A
MG1655 CspC-TGA-YFP-1 This study N/A
MG1655 CspC-TGA-YFP-2 This study N/A
BW25113 Drmf (Baba et al., 2006) N/A
MG1655 DprfC This study N/A
MG1655 attB::Ptet-m-y This study N/A
MG1655 attB::Ptet-m-TGA-y This study N/A
MG1655 D2 (DrrnEG) (Quan et al., 2015) N/A
MG1655 D4 (DrrnGBAD) (Quan et al., 2015) N/A
MG1655 D6 (DrrnGADBHC) (Quan et al., 2015) N/A
MG1655 prfB TAGC This study N/A
MG1655 prfB::3 3 FLAG This study N/A
CR201 N. De Lay N/A
Chemicals, Peptides, and Recombinant Proteins
Chloramphenicol Sigma Cat# C0378
ULTRAhyb Ultrasensitive Hybridization Buffer Invitrogen Cat# AM8670
Rifampicin CHEM-IMPEXINTL INC Cat# 00260
Erythromycin CHEM-IMPEXINTL INC Cat#00126
Tetracycline hydrochloride CHEM-IMPEXINTL INC Cat#00667
Spectinomycin dihydrochloride pentahydrate Sigma Cat# S9007
Critical Commercial Assays
iScript cDNA Synthesis Kit Bio-Rad Cat# 170-8891
SsoAdvanced Universal SYBR Green Supermix Bio-Rad Cat# 172-5271
In-Fusion HD Cloning Plus Takara Bio USA, Inc Cat# 638909
Q5 Hot Start High-Fidelity 2 3 Master Mix NEB Cat# M0494S
TRIzol Reagent Ambion Cat# 15596018
Experimental Models: Organisms/Strains
E. coli: K-12 MG1655 F#; l#; rph-1 E. coli Genetic Stock Center at Yale CGSC# 6300
E. coli: BL21 (DE3) Laboratory strain N/A
S. enterica serovar Typhimurium LT2 ATCC ATCC# 700720
Oligonucleotides
See Table S4 for full list of oligonucleotides used in This study N/A
this study
Recombinant DNA
pZS-Ptet -m-y This study N/A
pZS- Ptet -m-TGA-y This study N/A
pZS- Ptet -m-TAG-y This study N/A
(Continued on next page)

Molecular Cell 67, 826836.e1e5, September 7, 2017 e1

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
pZS- Ptet -m-y +1 fs This study N/A
pZS- Ptet -m-y #1 fs This study N/A
pZS*11-yfp13 (Subramaniam et al., 2013) N/A
ASKA-prfB (Kitagawa et al., 2005) N/A
pKD46 (Datsenko and Wanner, 2000) N/A
pCP20 (Datsenko and Wanner, 2000) N/A
pSIM6 (Datta et al., 2006) N/A
pKT-tRNASer_GCT (wild-type control) This study N/A
pKT-tRNASer_TCA (UGA suppressor) This study N/A
pZS- Pcp25 -Mut SD-m-y This study N/A
pZS- Pcp25 -Mut SD-m-TGA-y This study N/A
pZS- Pcp25 -Non-opt-m-y This study N/A
pZS- Pcp25 -Non-opt-m-TGA-y This study N/A
Software and Algorithms
MicrobeTracker 0.937 (Sliusarenko et al., 2011) N/A
Slidebook 6.0 3i Intelligent Imaging Innovation N/A
ImageJ 1.47v bundled with 32-bit Java 1.6.0_20 NIH N/A
Image Lab 6.0 for Windows Bio-Rad N/A
MATLAB R2012b 8.0.0.783 The MathWorks N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for reagents may be directed to, and will be fulfilled by the lead contact Jiqiang Ling (Jiqiang.Ling@
uth.tmc.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Bacterial Strains, Plasmids and Growth Conditions

All the bacterial strains and main plasmids used in this study are listed in the Key Resources Table. All the oligos for making the mutant
strains and plasmids are listed in Table S4. The mutant strains are derivatives of E. coli K-12 MG1655 (WT). To construct MG1655
CspC-TGA-YFP-1, MG1655 CspC-TGA-YFP-2, MG1655 attB::Ptet-m-y, MG1655 attB::Ptet-m-TGA-y, and MG1655 prfB::3 3
FLAG strains, a cassette (kan-ccdB) containing the toxin encoding gene ccdB under the control of araBAD promoter and a kanamycin
resistance gene was amplified from template genomic DNA of CR201 strain (obtained from N. De Lay, UTHealth), and introduced into
the specific sites on the chromosome of the parental strain harboring plasmid pSIM6 by l red recombinase-mediated gene replace-
ment (Datta et al., 2006). Strains containing pSIM6 were induced for Red expression by growth at 42! C for 15, and electroporated
with PCR fragments containing the kan-ccdB cassette. 1 mL LB was then added and cells were incubated at 32! C for 2 hr. The suc-
cessful transformants were selected by kanamycin resistance. The kan-ccdB cassette was then replaced with respective DNA frag-
ments. The positive clones were selected by growth on 0.5% arabinose LB plate, and verified by colony polymerase chain reaction
(PCR). The MG1655 prfB TAGC strain was generated with a modified multiplex automated genome engineering (MAGE) method
(Wang et al., 2009). Briefly, prfB-MAGE-TAGC oligo was electroporated into the MG1655 strain carrying plasmid pKD46, and cells
were plated onto LB plates after 5 hr incubation at 32! C. Colony PCR was applied with primer pairs prfB-TAGC-MT-F and prfB-
TAGC-R0 to select for the positive clones. All mutant strains were verified by sequencing.
To construct plasmids pZS-Ptet-m-y, pZS-Ptet-m-TGA-y, pZS-Ptet-m-TAG-y, pZS-Ptet-m-y +1 fs, and pZS-Ptet-m-y #1 fs, the pZS-
Ptet-29AA-y plasmid was generated first by ligating a 29 amino acid (LQTSAGEAAAKEAAAKEAAAKEAAAKAAA) linker with plasmid
pZS*11-yfp13 using In-Fusion HD Cloning according to manufacturers protocol. Fragments amplified from optimized mCherry
gBlock by primer pZS-mCherry-29AA-IF paired with pZS-mCherry-29AA-IR, pZS-mCherryTGA-29AA-IR, pZS-mCherryTAA-
29AA-IR, pZS-mCherryTAG-29AA-IR, pZS-mCherryTAAA-29AA-IR, and pZS-mCherryTA-29AA-IR, respectively, were ligated into
the pZS- Ptet-29AA-Y plasmid through In-Fusion HD Cloning.
Unless otherwise noted, E. coli strains were grown in LB at 37! C. For fluorescence microscopy analysis, overnight cultures were
diluted 1: 1,000 and grown for 24 hr. 100 mg/ml Chl was added to immediately stop protein synthesis. After 4 hr incubation to allow full
maturation of mCherry and YFP, samples were subjected to fluorescence microscopy analysis. The minimal medium contains

e2 Molecular Cell 67, 826836.e1e5, September 7, 2017

47.8 mM Na2PO4, 22.0 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 4 mM MgSO4, 0.2 mM CaCl2, 40 mg/ml each of the 20 amino
acids, and 0.4%1% glucose or indicated sugar.

METHOD DETAILS

Fluorescence Microscopy
Samples were placed on a 2 mL agarose (1.5%) phosphate buffer pad on 15-well Multitest Slides (MP Biomedicals, LLC.). Images
were obtained on an Olympus IX81-ZDC inverted microscope using Slidebook imaging software. Background fluorescence was sub-
tracted from each image using ImageJ Background Subtraction with a 50.0 pixel rolling ball radius. Single-cell fluorescence quan-
titation was completed with MicrobeTracker (Sliusarenko et al., 2011), a MATLAB-based software package. Single-cell fluorescence
data were exported from MATLAB and all further data analysis was completed in Microsoft Excel.

Time-lapse Microscopy
Overnight cultures were diluted 1:1,000 in LB and grown for 24 hr at 37! C. Cultures were placed on a 200 mL 1.5% agarose LB pad.
Fluorescent images were taken at the initial time point for quantitation. Cells were followed for 150 min at room temperature with DIC
images taken at regularly spaced intervals throughout the experiment. Image analysis and editing were performed using ImageJ.

Rate Determination
E. coli cultures were incubated at 37! C in a microplate reader (Synergy HT, BioTek) using 96-well black side plates (Corning). The
signals of mCherry, YFP, and A600 were measured every 20 min with fluorescence spectrometry. To calculate translational error
rates, the YFP/mCherry ratio of m-TGA-y, m-TAG-y, m-y +1 fs, and m-y #1 fs was normalized by the YFP/mCherry ratio of the control
m-y reporter. Protein synthesis rates were calculated as described (Subramaniam et al., 2013) with the following formula:
Protein synthesis rate; S = 1=Absorbance3dfluorescence=dtime.
To calculate translational error rates by western blotting, E. coli strains were grown in LB medium with and without addition of chlor-
amphenicol at 37! C for 24 hr before 10 mL of culture was harvested. The cells were lysed by sonication, and whole protein samples
were analyzed by western blotting with a primary antibody against mCherry. The signals were qualified by Image Lab (Bio-Rad), and
the error rates were calculated as the percentage of mCherry-YFP fusion protein.

Calculation of CV and Noise

CV was calculated as the standard deviation (s) divided by the mean (m) of the YFP/mCherry ratio of each cell in the same microscopic
image frame. Noise was calculated as the ratio of the variance (s2) over the square of the mean (m2).

Detection of Native UGA Readthrough in cspC

The readthrough level of cspC UGA was determined by western blotting. E. coli strains were grown in LB with and without addition of
Chl (2 mg/ml) at 37! C for 24 hr. 10 mL of cell culture were harvested and lysed by sonication, and total proteins were separated by
SDS-PAGE. Antibodies against GFP (Roche) and RpoB (loading control, Santa Cruz) were used in western blotting analysis. The sig-
nals were qualified by Image Lab (Bio-Rad).

RNA Isolation
To prepare total RNA for qRT-PCR, cells in LB medium with or without 10 mM IPTG were grown to mid-log phase (OD600"0.6-0.8).
800 mL of culture was used for total RNA extraction using hot phenol, and residual chromosomal DNA was removed.
To isolate total RNA for acidic gel Northern blotting, overnight cultures were diluted 1: 1,000 in LB and grown for 14 hr at 37! C.
10 mL of cell culture was harvested at 4! C and resuspend with TRIzol reagent immediately. Cells were lysed by the beads beater
with 0.1 mm glass beads (RPI). RNA sample was prepared according to manufacturers protocol, and was stored in 10 mM sodium
acetate buffer (pH 5.0) with 1 mM ethylenediaminetetraacetic acid (EDTA) at #80! C to preserve aminoacyl-tRNAs. Deacylated sam-
ples were obtained by incubating RNA in 200 mM Tris pH 9.0 at 37! C for 30 min.

qRT-PCR
1 mg of total DNA-free RNA was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad) according to manufacturers protocol.
cDNA was amplified with the corresponding primers (see Table S4). qPCR was performed using Bio-Rad CFX96 and SsoAdvanced
Universal SYBR Green Supermix (Bio-Rad) according to manufacturers suggestion. mreB transcript level was used for normaliza-
tion. The DDCt method was used to obtain the fold changes of target genes.

Acidic Gel Northern Blotting

Acid urea polyacrylamide gel electrophoresis was performed according to the procedures described in (Janssen et al., 2012). Briefly,
12% Acid urea polyacrylamide gel was prepared freshly before use. 5 mg of RNA sample was loaded onto the gel, and subjected the
electrophoresis with sodium acetate running buffer (100 mM sodium acetate pH5.0, 1 mM EDTA) in cold room for 6 hr. Gel-separated
RNA was transferred to GT membrane (Bio-Rad) by semi-dry electro-blotting at 15 V for 40 min in 0.5 3 TBE. The membrane was

Molecular Cell 67, 826836.e1e5, September 7, 2017 e3

cross-linked by UV and probed with 50 end biotin labeled DNA oligonucleotide probes (see Table S4). Trp-tRNATrp, tRNATrp, Gly-
tRNAGly and tRNAGly were detected by the Northern blotting, respectively, with SsrA as the loading control. Quantitation was per-
formed with Image Lab (Bio-Rad).

Protein Digestion and Proteomics

Protein extraction and digestion from E. coli was performed using cell pellets from 200 mL stationary-phase cultures. Lysis, reduction,
alkylation, trypsin digest, and acid cleavage were performed as in (Lajoie et al., 2013). All resulting peptides were purified and de-
salted using a C18 MacroSpin column (The Nest Group), dried in a rotary vacuum centrifuge, and resuspended in 6 mL 70% formic
acid and 16 mL 0.1% trifluoroacetic acid. Following A280 peptide quantification, samples were diluted to a concentration of 0.5 mg/mL
in the same buffer, and 4 mL of sample (2 mg total) were injected onto an analytical column using ACQUITY UPLC M-Class (Waters) for
mass spectrometry. Column specifications, solvent gradients, and mass spectrometry parameters for the Q Exactive Plus (Thermo)
were performed as described in (Ferdaus et al., 2016).

Peptide Database Construction and Searches

The MG1655 genome and annotated open reading frames (ORFs) were downloaded from https://www.ncbi.nlm.nih.gov/ on
September 16th, 2016. ORFs terminating in TGA were mapped to the reference genome and extended to the next in-frame stop
codon (TAG, TAA or TGA) using an ad hoc Python script. Extended ORFs were translated and separate entries were included in
the search database for every natural amino acid or an in-frame skipping event at the first TGA position. The normal MG1655 pro-
teome and the extended frame protein sequences (sequences beginning at the first tryptic residue N-terminal to the TGA), including
the m-TGA-y reporter sequences, were used concurrently for mass spectrometry data searches using MaxQuant v.1.5.1.2 (Cox and
Mann, 2008). The copy number of mCherry was estimated using Intensity Based Absolute Quantitation as described (Soufi et al.,
2015). The concentration of total mCherry was similar to that of EF-Tu corresponding to "300,000 molecules per cell. The
mCherry-YFP fusion protein was "2% of total mCherry as observed by fluorescence spectrometry and western blotting ("6,000 mol-
ecules per cell).

Mathematical Modeling
Theoretical modeling of the variation of stop codon (UGA) read-through error with antibiotic chloramphenicol (Chl) concentration are
based on the network shown in Figures S4B and S4C. In the scheme, E represents the bare ribosome with UGA mRNA stop codon in
A-site, ER represents the release factor (RF2)-bound state that undergoes a conformational transition to state ER* and PR is the state
generated after RF2-induced pH-dependent hydrolysis of the peptide chain in the ribosome P-site (Indrisiunaite et al., 2015). Here,
RF2 is the right substrate. For the wrong substrate, i.e., the ternary complex (TC) of Trp-tRNATrp, the initial bound state ET undergoes
GTP-hydrolysis to ET*. From state ET*, the system can take two routes: (i) it can accommodate the aminoacyl-tRNA that leads to
peptide chain elongation, i.e., readthrough of the stop codon (state PT) or (ii) it can reset by a proofreading dissociation of the aa-
tRNA from ribosome (Cochella and Green, 2005). No such proofreading appears to be present for the RF2 pathway (Freistroffer
et al., 2000).
A recent study shows that although Chl reduces the overall growth rate of E. coli, the elongation rate actually rises with Chl con-
centration (Dai et al., 2016). This feature is attributed to an increase in RNA/protein ratio that leads to a rise in effective TC concen-
tration. Our experiment also confirms this fact with the Trp-tRNATrp levels going up with [Chl] (see Figure 4). In contrast, the protein
levels go down with [Chl] (Dai et al., 2016). In addition, binding of Chl to ribosome stalls the translation process and this leads to a fall in
the completion probability of translation. We model these three effects of antibiotic addition with the following three schemes.
Model 1
Here, we take the TC concentration to be an increasing function of Chl concentration as
! "
Chl'
TC' = TC'0 1 + f (1)
Chl' + a
Here, [TC]0 denotes TC concentration in absence of Chl. We assume [TC] to be proportional to the Trp-tRNATrp level and fit the
experimentally obtained fold change using Equation (1). This gives f = 20:2; a = 18:6 g/ml. Now, our experimentally measured growth
rate in absence of Chl is "1.9 h#1. With this information and following a recent analysis of TC concentration for various tRNA species
under different growth conditions (Rudorf and Lipowsky, 2015), we set [TC]0 = 3.2 mM. We do not consider here any dependence of
[RF2] on [Chl]. We take [RF2]0 = 18 mM following a report of RF2 level variation at various E. coli growth rates (Adamski et al., 1994)
Also, in this model, we assume the completion probability to be 100%, i.e., all translation initiation events go to completion.
Model 2
Here, [TC] follows Equation (1). The [RF2] dependence on [Chl] is taken as
#1
RF2' = RF2'0 1 + KChl' (2)

e4 Molecular Cell 67, 826836.e1e5, September 7, 2017

 The parameter K ("0.4 mM#1) (Dai et al., 2016) is the binding constant of Chl with ribosome. This corresponds to the assumption
that decrease in the fraction of active ribosome leads to proportional decrease in translation rates and correspondingly in the protein
concentrations. Again, we take the completion probability to be 100%.
Model 3
In this case, the [TC] and [RF2] dependences on [Chl] are given by Equation (1) and Equation (2), respectively. However, we now note
that not every read-through even will result in the completed (functional) YFP protein. Therefore, if the translating ribosome binds Chl
before YFP is completed we assume that the translation will be aborted and the read-through wont result in YFP fluorescence signal.
The resulting functional dependence of completion probability on Chl is modeled as
T #1
Pcom = (3)
T #1 + kb Chl'
Here, T is the completion time of YFP protein synthesis, indicator of stop codon readthrough error. It is defined as N=r where
N = 251 is the number of amino acids (aa) in YFP and r is the elongation rate (Dai et al., 2016). We take r = 17 aa/s based on the values
reported in (Dai et al., 2016). The effective binding constant kb "2 X 104 M#1.s#1 is taken from a recent study on Chl inhibition
(Xaplanteri et al., 2003) that indicates that initial binding of Chl is fast followed by a slower isomerization step.
We study the kinetics of the whole network using the standard tools of first-passage technique (Kampen, 2007). The probability to
reach a given end-state (PR or PT) before the other, starting from state-E, is called the splitting probability (9). The UGA read-through
error is defined as the ratio of the splitting probability of reaching the wrong end (PT) to that of reaching the right end (PR). The param-
eter set to generate the nature of error variation as a function of Chl concentration are given in Table S2.

QUANTITATION AND STATISTICAL ANALYSES

Statistical parameters for each experiment are reported in the corresponding figures. All data presented are the mean of at least three
repeats, with error bars showing one standard deviation. The p values were calculated with unpaired t tests.

Molecular Cell 67, 826836.e1e5, September 7, 2017 e5