1. Genetics and Genomics
  2. Microbiology and Infectious Disease
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Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue

  1. Natalie Jing Ma
  2. Colin F Hemez
  3. Karl W Barber
  4. Jesse Rinehart  Is a corresponding author
  5. Farren J Isaacs  Is a corresponding author
  1. Yale University, United States
  2. Yale University School of Medicine, United States
Research Article
Cite this article as: eLife 2018;7:e34878 doi: 10.7554/eLife.34878
4 figures, 3 tables and 6 additional files

Figures

A UAG-ending transcript in the genomically recoded organism (GRO) may produce proteins with multiple differing C-termini.

(A) Unassigned codons arise when either the cognate tRNA or release factor recognizing a codon are removed. (B) Since the GRO lacks Release Factor 1 (RF1), ribosomal stalling at the UAG codons results in three possible fates for the nascent protein (blue): (1) suppression of the codon by a near-cognate or suppressor tRNA (yellow) and continued translation, (2) frameshifting of bases along the mRNA transcript into a new reading frame and continued translation (purple), or (3) ribosomal rescue by the ssrA-encoded tmRNA, ArfA, or ArfB proteins. If ribosomal rescue occurs via tmRNA, the resulting protein is tagged with a peptide sequence (red) for degradation, while rescue via ArfA or ArfB results in release of peptide without C-terminal modification.

https://doi.org/10.7554/eLife.34878.003
UAG codons in the genomically recoded organism elicit suppression, frameshifting, and tagging for degradation by the tmRNA.

(A) Schematic of the GFP construct with a C-terminal 6x-His tag and a UAG stop codon, showing 102 nucleotides downstream of the UAG codon and the positions of other stop codons in the downstream tail. (B) Peptides identified from the C-terminus of a UAG-ending GFP construct expressed in the GRO (using libraries detailed in Supplementary file 3 and 4). Purified GFP protein was digested with trypsin, processed via MS/MS, and the resulting data were computationally searched using libraries encoding all possible suppressors and all possible subsequent reading frames. Peptides are mapped to the C-terminus of the original GFP construct and grouped by reading frame, with the number of bases skipped listed in the left column. Green text represents GFP, blue text represents the C-terminal 6xHis tag and unframeshifted readthrough, orange text represents the position of a UAG stop codon, purple text represents frameshifted readthrough, and red text represents the tmRNA tag. Black dashes represent ribosomal frameshifts (Figure 2—source datas 1 and 2). (C) MS-MS spectra for two peptides: the C-terminus of GFP with the appended degradation tag (LEHHHHHHAANDENYALDD) and the C-terminus of GFP demonstrating a + 10 base skip in translation (LEHHHHHHGDPMVR). The other spectra validated from UAG-GFP expressing GRO.AA are shown in Supplementary file 2.

https://doi.org/10.7554/eLife.34878.004
Figure 2—source data 1

Raw data and analysis of peptides detected in mass spectrometry datasets using a library generated to search for frameshifting, near-cognate suppression, and ribosomal rescue events (Supplementary file 3).

https://doi.org/10.7554/eLife.34878.005
Figure 2—source data 2

Raw data and analysis of peptides detected in mass spectrometry datasets using a library generated to search for loss of translational fidelity (Supplementary file 4).

https://doi.org/10.7554/eLife.34878.006
Figure 3 with 1 supplement
Deletion of both ssrA and arfB restores protein production in the genomically recoded organism.

(A) Comparison of doubling times for WT and GRO strains carrying listed deletions with and without GFP induction. Error bars show standard deviation centered at mean, n = 3; data were analyzed using Source code 1 (Figure 3—source datas 1 and 2). (B) Change in maximum optical density at 600 nm (OD600) due to expression of UAG-GFP or UAA-GFP in wild-type (WT) and GRO strains carrying listed deletions. Error bars show standard deviation centered at mean, n = 3 (Figure 3—source datas 1 and 2). (C) Quantification of GFP abundance per 1 mL of cells at OD600 of 2.5 via western blot from biological replicates of indicated strains (Figure 3—source datas 36). Error bars show standard deviation centered at mean, n = 3 (Figure 3—source datas 35). See Figure 3—figure supplement 1 for linear calibration curves used to quantify GFP abundance for each replicate experiment. Image of representative western blot is below the graph. p-values are calculated in relation to the GRO containing the UAG-ending GFP (GRO – UAG) and are as follows: * is p≤0.05, ** is p≤0.01, *** is p≤0.001, and **** is p≤0.0001.

https://doi.org/10.7554/eLife.34878.009
Figure 3—source data 1

Growth curve data from 96-well plate assay analyzed using Source code 1 (one of three plate replicates), used for data represented in Figure 3A and B.

https://doi.org/10.7554/eLife.34878.011
Figure 3—source data 2

Analysis of doubling times and maximum OD600’s of indicated strains.

File contains doubling times and maximum OD600’s for three separate experiments conducted on different plate reader machines. Each experiment tested each sample in biological triplicate. Only the biological triplicate data from Plate 3 is represented in Figure 3A and B.

https://doi.org/10.7554/eLife.34878.012
Figure 3—source data 3

Anti-GFP western blot image used for quantification of GFP yields; replicate 1.

https://doi.org/10.7554/eLife.34878.013
Figure 3—source data 4

Anti-GFP western blot image used for quantification of GFP yields; replicate 2.

https://doi.org/10.7554/eLife.34878.014
Figure 3—source data 5

Anti-GFP western blot image used for quantification of GFP yields; replicate 3.

https://doi.org/10.7554/eLife.34878.015
Figure 3—source data 6

Analysis of western blot data represented in Figure 3C.

https://doi.org/10.7554/eLife.34878.016
Figure 3—figure supplement 1
Calibration curves used for quantification of GFP yields, as represented in Figure 3C, using GFP samples of known concentration.

Replicate 1 corresponds to the western blot shown in Figure 3—source data 3; Replicate 2 corresponds to the western blot shown in Figure 3—source data 4; Replicate 3 corresponds to the western blot shown in Figure 3—source data 5.

https://doi.org/10.7554/eLife.34878.010
Deleting ssrA restores propagation of both viruses and conjugative plasmids in the genomically recoded organism.

(A) Percent transfer of conjugative plasmid RK2 from a wild-type donor into wild-type (WT), GRO, or GRO with designated deletions (KO) as recipients (Figure 4—source data 1). Data are obtained from technical triplicates generated from a single biological sample. (B) Percent increase in doubling time for strains carrying plasmid RK2 compared to strains lacking RK2 (Figure 4—source datas 2 and 3). (C) Number of conjugation events for conjugative plasmid F from wild-type, GRO, or GRO with designated gene deletions as donors to a wild-type recipient (Figure 4—source data 4). Data are obtained from technical triplicates generated from a single biological sample. (D) Relative titer on wild-type, GRO, and GRO with designated deletions of phage λ (Figure 4—source data 5). Error bars show standard deviation centered at mean, n = 3. p-values are calculated in relation to the GRO condition and are as follows: * is p≤0.05, ** is p≤0.01, *** is p≤0.001, and **** is p≤0.0001. (E) Effects of sequential deletions of ribosomal rescue mechanisms on conjugative plasmid transfer efficiency. (F) Effects of sequential deletions of ribosomal rescue mechanisms on viral susceptibility.

https://doi.org/10.7554/eLife.34878.017
Figure 4—source data 1

Analysis of RK2 plasmid conjugation data represented in Figure 4A.

Note: These data represent technical triplicates generated from the same biological sample.

https://doi.org/10.7554/eLife.34878.018
Figure 4—source data 2

Growth curve data from 96-well plate assay analyzed using Source code 1, used for data represented in Figure 4B.

https://doi.org/10.7554/eLife.34878.019
Figure 4—source data 3

Analysis of doubling times represented in Figure 4B.

https://doi.org/10.7554/eLife.34878.020
Figure 4—source data 4

Analysis of F plasmid conjugation data represented in Figure 4C.

Note: These data represent technical triplicates generated from the same biological sample.

https://doi.org/10.7554/eLife.34878.021
Figure 4—source data 5

Analysis of lambda phage infection data represented in Figure 4D.

https://doi.org/10.7554/eLife.34878.022

Tables

Table 1
Strains used in this study.
https://doi.org/10.7554/eLife.34878.007
Strain Abbreviation*Ancestor (source)Genotype# UAG CodonsRF1 Status§Ribosomal rescue gene deletionssrA tag Status#Investigated in
GRO.DD.prfA+GRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla]0+RF1n/aDDGFP expression for mass spectrometry (Figure 2)
GRO.DDGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1n/aDDGFP expression for mass spectrometry (Figure 2)
ECNR2.AAE. coli MG1655 (Wang et al., 2009)MG1655 ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla]321+RF1n/aAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AAECNR2.AA (Lajoie et al., 2013b)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1n/aAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AA.∆ssrAGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1ssrAAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AA.∆arfAGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1arfAAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AA.∆arfBGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1arfBAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AA.∆ssrA.arfBGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1ssrA,arfBAAFitness, conjugation, and viral infection (Figures 3 and 4)
GRO.AA.∆arfA.arfBGRO.AA (this study)ΔmutS:zeo.Δ(ybhB-bioAB):[λcI857.Δ(cro-ea59):tetR-bla], ΔprfA, ΔtolC0∆RF1arfA,arfBAAFitness, conjugation, and viral infection (Figures 3 and 4)
  1. *All strains derived from ECNR2, as described in Wang et al. (2009).

    †See Key Resources Table for additional information on strains and sources. The GenBank accession number for E. coli MG1655 is U00096, and the GenBank accession number for GRO.AA is CP006698.

  2. ‡ Out of a total of 321 in the original ECNR2 strain.

    §RF1 terminates translation at UAG and UAA. Deletion of RF1 eliminates recognition of UAG during translation; translational termination continues through RF2, which recognizes UAA and UGA.

  3. #The ssrA gene encodes the tmRNA, which appends the ssrA degradation tag to stalled ribosomes. The wild-type sequence is AANDENYALAA; mutation of the C-terminus to AANDENYALDD slows degradation of peptides to enable detection by mass spectrometry.

Table 2
Components of peptide library constructed to search and analyze tandem mass spectrometry data.

The LEHHHHHHXXX library was separate from the library that contained the entries of the first three rows of the table (see Supplementary file 3 and 4).

https://doi.org/10.7554/eLife.34878.008
Library componentExample peptides (from Figure 2A)Enables detection of…Complete peptide list
Any one of 20 canonical amino acids inserted at the UAG codonLEHHHHHHQGARNear-cognate suppressionSupplementary file 3
Any length of C-tail following UAG codon to the next non-UAG stop codon or to 38 amino acids downstream of the UAG codon, whichever came firstALGDPMVRReadthrough, frameshifting, and rescue by ArfA or ArfBSupplementary file 3
AANDENYALDD degradation tagLEHHHHHHGDAANDENYALDDRescue by tmRNA-SmpBSupplementary file 3
All peptides of form LEHHHHHHXXX, where X is any amino acidLEHHHHHHQLDLoss of translational fidelitySupplementary file 4
Key resources table
Genetic reagents, bacterial strains, antibodies, and software used in this study.
https://doi.org/10.7554/eLife.34878.023
Reagent
type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Isaacs
Lab
Reference
#
Full
genotype
of strains
# UAG
Codons
RF1
status
Ribosomal
rescue
gene
knockout
ssrA
tag
status
Gene
(Escherichia
coli)
pUAG-GFPthis papereGFP-6xHis
-UAG; Plasmid
NJM88;
Strain
NJM1242
eGFP protein
with a C-terminal
6-His tag for protein
purification,
terminating
translation in a
UAG codon.
Plasmid
NJM88;
Strain
NJM1242
N/AN/AN/AN/AN/A
Gene
(E. coli)
pUAA-GFPthis papereGFP-6xHis
-UAA; Plasmid
NJM89;
Strain
NJM1249
eGFP protein with
a C-terminal 6-His
tag for protein
purification,
terminating
translation in a
UAA codon.
Plasmid
NJM89;
Strain
NJM1249
N/AN/AN/AN/AN/A
Genetic
reagent
(E. coli)
RK2410.1126/science
.1205822;
10.1016/j.cels
.2016.06.009
pRK24;
Strain NJM699
Conjugative RK2
plasmid (10.1006/
jmbi.1994.1404),
but lacks functional
AmpR gene.
Strain
NJM699
N/AN/AN/AN/AN/A
Genetic
reagent
(E. coli)
FYale University
Coli Genetic
Stock Center
(CGSC),
Strain #4401
pF; Strain
EMG2; Strain
CGSC#4401;
Strain
NJM426;
Strain
NJM473
Conjugative F
plasmid, as
described by
PMID: 4568763.
Obtained from
the Yale CGSC.
Strain
NJM426;
Strain
NJM473
N/AN/AN/AN/AN/A
Genetic
reagent
(E. coli)
pZE21_
UAG-GFP
this paperpZEtR-eGFP
-cHis-TAG-
v02; Plasmid
NJM88;
Strain
NJM1242
pZE21 plasmid
with pLtetO
promoter driving
inducible expression
of eGFP with a
C-terminal 6-His
tag and terminating
in UAG codon.
Inducible with
anhydro-tetracycline.
Plasmid
NJM88;
Strain
NJM1242
N/AN/AN/AN/AN/A
Genetic
reagent
(E. coli)
pZE21_
UAA-GFP
this paperpZEtR-eGFP
-cHis-TAA-v02
; Plasmid
NJM89;
Strain
NJM1249
pZE21 plasmid
with pLtetO
promoter driving
inducible expression
of eGFP with a
C-terminal 6-His
tag and terminating
in UAA codon.
Inducible with
anhydro-tetracy
cline.
Plasmid
NJM89;
Strain
NJM1249
N/AN/AN/AN/AN/A
Genetic
reagent
(Enteroba
cteria
phage λ)
λ.CI857Coli Genetic
Stock Center
(CGSC), Yale
University
(contact John
Wertz directly)
λ.CI857; λ
phage;
Phage NJM102
Phage λ with
temperature-
sensitive CI
repressor gene;
when incubated
at 37° C, phage
becomes obligate
lytic
Phage
NJM102
N/AN/AN/AN/AN/A
Cell line
(E.
coli)
GRO.DDthis paperC31GIB.
tmRNA-DD;
Strain #987
MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and tmRNA tag
C-terminal amino
acids mutated from
AA to DD. Retains
lambda red cassette
for recombineering.
Investigated in
Figure 2.
Strain
#987
ΔmutS:zeo.
Δ(ybhB-
bioAB)
:[λcI857.
Δ(cro-ea59)
:tetR-bla].
ΔprfA.ΔtolC
.tmRNADD
0+RF1n/aDD
Cell line
(E. coli)
GRO.
DD.prfA+
this paperC31GIB.
prfA+.tmRNA
-DD; Strain
#996
MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
retains RF1,
and tmRNA tag
C-terminal amino
acids mutated from
AA to DD. Retains
lambda red cassette
for recombineering.
Investigated in
Figure 2.
Strain
#996
ΔmutS:zeo.
Δ(ybhB-
bioAB)
:[λcI857.
Δ(cro-ea59):
tetR-bla].
ΔtolC.tm
RNADD
0∆RF1n/aDD
Cell line
(E. coli)
ECNR210.1016/j.cels
.2016.06.009
ECNR2.Δmut
S:zeocin.Δ
λRed; Strain
#795
MG1655-derived
strain that contains
321 UAG codons
and retains RF1.
Investigated in
Figures 3 and 4.
Strain
#795
ΔmutS:zeo321+RF1n/aAA
Cell line
(E. coli)
GRO.AA10.1016/j.cels
.2016.06.009
C31.final.
ΔmutS:
zeocin.ΔprfA
.ΔλRed;
Strain #796
MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1.
Investigated in
Figures 3 and 4.
Strain
#796
ΔmutS:
zeo.ΔprfA
(GenBank
ID:
CP006698)
0∆RF1n/aAA
Cell line
(E. coli)
GRO.
AA.∆arfB
this paperC31GIB.arfB:
tolCorf.
ΔλRed;
Strain #1230
MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and deletion of arfB.
Investigated in
Figures 3 and 4.
Strain
#1230
ΔmutS:
zeo.ΔprfA
.arfB:tolC
0∆RF1∆ssrAAA
Cell line
(E. coli)
GRO.
AA.∆ssrA
this paperC31GIB.ssrA
:tolC.ΔλRed;
Strain #1231
MG1655-derived
strain with all 321
UAG codons
mutated to UAA,
deletion of RF1,
and deletion
of ssrA.
Investigated in
Figures 3 and 4.
Strain
#1231
ΔmutS:
zeo.ΔprfA.
ssrA:tolC
0∆RF1∆arfAAA
Cell line
(E. coli)
GRO.
AA.∆arfA
this paperC31GIB.arfA
:tolC.ΔλRed
; Strain #1232
MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
arfA. Investigated
in Figures 3 and 4.
Strain
#1232
ΔmutS:
zeo.ΔprfA.
arfA:tolC
0∆RF1∆arfBAA
Cell line
(E. coli)
GRO.AA
.∆ssrA.∆arfB
this paperC31GIB.ΔarfB
.ssrA:tolC.Δ
λRed; Strain
#1233
MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
ssrA and arfB.
Investigated in
Figures 3 and 4.
Strain
#1233
ΔmutS:
zeo.ΔprfA
.ΔarfB.ssrA:tolC
0∆RF1∆ssrA.
∆arfB
AA
Cell line
(E. coli)
GRO.AA
.∆arfA.
∆arfB
this paperC31GIB.Δarf
B.arfA:tolC.
ΔλRed;
Strain #1234
MG1655-derived
strain with all
321 UAG codons
mutated to UAA,
deletion of RF1,
and deletion of
arfA and arfB.
Investigated in
Figures 3 and 4.
Strain
#1234
ΔmutS:
zeo.ΔprfA
.ΔarfB.arfA
:tolC
0∆RF1∆arfA.
∆arfB
AA
Antibodymouse
anti-GFP
antibody
otherInvitrogen
(Ref#: 332600,
Lot#:
1513862A)
Invitrogen
(Ref#: 332600,
Lot#: 1513862A);
(5.5 μL antibody
in 3 mL Milk
 + TBST)
N/AN/AN/AN/AN/AN/A
Antibodygoat
anti-mouse
antibody
otherAbCam (Ref#:
ab7023, Lot#:
GR157827-1)
AbCam (Ref#:
ab7023, Lot#:
GR157827-1);
(2.2 μL antibody
in 10 mL Milk
 + TBST)
N/AN/AN/AN/AN/AN/A
Recombinant
DNA reagent
ssrA:tolCthis paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081
dsDNA
NJM111
The E. coli native
tolC gene used to
delete ssrA gene via
recombineering
(10.1038/nprot.
2008.227).
dsDNA
NJM111
N/AN/AN/AN/AN/A
Recombinant
DNA reagent
arfA:tolCthis paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081
dsDNA
NJM112
The E. coli native
tolC gene used to
delete arfA gene
via recombineering
(10.1038/nprot.
2008.227).
dsDNA
NJM112
N/AN/AN/AN/AN/A
Recombinant
DNA reagent
arfB:tolCthis paper; for
use, see
tolC positive
/negative
selection in
10.1038/nprot
.2014.081
dsDNA
NJM113
The E. coli native
tolC gene used to
delete arfB gene
via recombineering
(10.1038/nprot.
2008.227).
dsDNA
NJM113
N/AN/AN/AN/AN/A
Software,
algorithm
Doubling
time
algorithm
10.1126/
science.1241459
Growth_
Analyze_
GK.m
Doubling time
used in 10.1126
/science.1241459,
written by Gleb
Kuznetsov in the
lab of Dr. George
Church.
N/AN/AN/AN/AN/AN/A
Software,
algorithm
MaxQuant
v1.5.1.2
otherN/ACommercial
software for
mass
spectrometry
analysis.
N/AN/AN/AN/AN/AN/A
Software,
algorithm
Graphpad
Prism 7
otherN/ACommercial
software for
statistical
analysis and
graphing,
provided
through Yale
University.
N/AN/AN/AN/AN/AN/A

Additional files

Source code 1

MATLAB script used to analyze growth curve data from 96-well plate assays (Figure 3A,B and 4B).

https://doi.org/10.7554/eLife.34878.024
Supplementary file 1

Gene nucleotide sequences, processed mass spectrometry data, and numerical values used to generate Figures 24.

https://doi.org/10.7554/eLife.34878.025
Supplementary file 2

Spectra for all 47 manually verified peptides detected through mass spectrometry from GRO.AA expressing the UAG-GFP plasmid.

https://doi.org/10.7554/eLife.34878.026
Supplementary file 3

Library of peptides generated for the detection of frameshifting, near-cognate suppression, and ribosomal rescue events from mass spectrometry data.

https://doi.org/10.7554/eLife.34878.027
Supplementary file 4

Library of peptides generated for the detection of loss of translational fidelity from mass spectrometry data.

https://doi.org/10.7554/eLife.34878.028
Transparent reporting form
https://doi.org/10.7554/eLife.34878.029

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