Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction Brian R. Fritz1, Osman K. Jamil1, and Michael C. Jewett1,2,3,4,5,* 1
Department of Chemical and Biological Engineering Interdisciplinary Biological Sciences Graduate Program, 3 Northwestern Institute on Complex Systems, 4 Institute for Bionanotechnology in Medicine, 5 Chemistry of Life Processes Institute, 2
Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA. * To whom correspondence should be addressed. Tel: +1 847 467 5007; Email:
[email protected]
Supplementary Information
Fritz et al., 2015
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SUPPLEMENTARY METHODS 14 C-leucine incorporation assay 14
C-leucine incorporation was used for general quantification of translation activity, independent
of reporter protein activity. L-[14C(U)]-Leucine (PerkinElmer Inc., NEC279E) was included at a concentration of 10 µM (50 nCi per reaction) in iSAT reactions containing one of the following reporter plasmids: pY71mRFP1, pY71sfGFP, or pK7Luc. Reactions were incubated at 37°C for 18 h, stopped by addition of 100 µL 0.1 N NaOH, and incubated at 37°C for an additional 20 min. 50 µL of each diluted reaction was pipetted on each of two Whatman 3MM chromatrography paper strips and dried. To precipitate polypeptides, one strip for each reaction was washed 3 times with 100 mL 5% trichloroacetic acid (TCA) and 1 time with 100 mL 100% ethanol, and dried again. Washed and unwashed sample strips were analyzed in a MicroBeta2 liquid scintillation counter (PerkinElmer Inc.) as a measure of precipitated and total radioactivity, respectively. Protein production was calculated using the following equation:
Protein!produced! !M = !
!"#$%&%'('#)!!"#!(!"#) !"#$%&%'('#)!!"#!(!"#) ! !"#$%!!"#!(!"#)! !"#$%!!"#!(!"#)! !"# !"#$
!".!!!"!!"#!!"#!!"#$!%"!!!"#$%&'
∗[!"#$%!!"#!!"!!!]
[1]
where background (‘bkgd’) reactions lacked reporter plasmids (Kim, et al. (1996) Eur. J. Biochem., 239, 881-886).
Proteomic analysis of r-proteins in 70S ribosomes
Ribosomal proteins were precipitated in 20% trichloroacetic acid (TCA) by incubation at 4°C overnight and centrifugation at 14,000 x g for 10 min. Precipitated proteins were washed with cold 10% TCA once and acetone twice. The protein pellet was air dried for 10-20 min prior to resuspension in 20 µL 8 M urea. Proteins were reduced with 10 mM dithiothreitol and cysteine residues alkylated with 50 mM iodoacetamide. Sequencing grade trypsin (Promega) was added at a 1:50 enzyme:protein ratio and after overnight digestion at room temperature, the reaction was stopped by addition of formic acid to 1%. Following digestion, peptides were desalted using C18 Spin columns (Pierce, cat # 89870) and lyophilized. Amino reactive TMT reagents (126/127, Thermo Scientific, cat# 90065) were dissolved in 41 µL acetonitrile and added to the lyophilized peptides
Fritz et al., 2015
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dissolved in 100 µL of 100 mM triethylammonium bicarbonate. After 1 h at room temperature, the reaction was quenched by adding 8 µL of 5% hydroxylamine. Following labeling, the two samples under analysis were mixed in a 1:1 ratio. Peptides were desalted using C18 ZipTip Pipette Tips (EMD Millipore) and resuspended in 30 µL of solvent A (95% water, 5% acetonitrile, 0.2% formic acid). Resuspended peptides (6 µL) were injected onto a trap column (150 µm ID × 3 cm) using an autosampler (Thermo Dionex). A nanobore analytical column (75 µm ID × 15 cm) was coupled to the trap in a vented tee setup. Upstream of the column, a 15 µm spray tip from New Objective was connected. The trap and analytical column were packed with Phenomenex Jupiter C18 media (5 µm). The Dionex Ultimate 3000 system was operated at a flow rate of 2.5 µL/min for loading onto the trap. Proteins were separated on the analytical column and eluted into the mass spectrometer using a flow rate of 300 nL/min and the following gradient: 2% B at 0 min.; 30% B at 85 min.; 55% B at 88 min.; 95% B from 90-93 min.; 2% B from 94 to 110 min. Solvent A consisted of 95% water, 5% acetonitrile, 0.2% formic acid and solvent B consisted of 5% water, 95% acetonitrile, 0.2% formic acid. Peptides were eluted into a nanoelectrospray ionization (nESI) source on an Orbitrap Elite mass spectrometer (Thermo Scientific). The MS method included the following events: 1) precursor scan, FT, m/z 400–1,600, 60,000 resolution and 2) datadependent MS/MS on the top 10 peaks in each spectrum from scan event 1 using higher-energy collision dissociation (HCD) with the following parameters: 15,000 resolution, precursor isolation width 2 m/z, NCE 35, activation time 100ms. The automatic gain control (AGC) settings were 1 × 106 ions and 5 × 104 ions for precursor and MS/MS scans, respectively. Singly-charged and ions for which a charge state could not be determined were not subjected to MS/MS. Proteome Discoverer (Thermo Scientific) and the Sequest algorithm was used for data analysis. Data was searched against a custom database containing UniProt entries using Escherichia coli taxonomy, allowing 3 missed cleavages, 10 ppm precursor tolerance, and carbamidomethylation of cysteine as a static modification. Variable modifications included oxidation of methionine, TMT of lysine and N-terminal TMT. For quantification via the reporter ions the intensity of the signal closest to the theoretical m/z, within a ±10 ppm window, was recorded.
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Reporter ion intensities were adjusted based on the overlap of isotopic envelopes of all reporter ions as recommended by the manufacturer. Only peptides with high confidence were used for quantification. Ratios of 126/127 were normalized based on median.
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Supplementary Figure S1
Supplementary Figure S1. iSAT protein synthesis of 3 different reporter proteins under crowding and reducing conditions. iSAT reactions with
14
C-leucine were performed with
pY71mRFP1, pY71sfGFP, or pK7Luc reporter constructs and 6% w/v Ficoll 400 and 2 mM DTBA, individually or in combination. Reactions were incubated at 37°C for 18 h, and protein synthesis was assessed by the incorporation of radioactive
14
C-leucine into precipitable
polypeptides (Supplementary Methods). Values represent average protein synthesis (n=3) relative to protein synthesis of reactions with no additives (where ‘1’ corresponds to: mRFP1 = 1.9 ± 0.3 µM, sfGFP = 3.4 ± 0.7 µM, Luc = 0.30 ± 0.04 µM) and error bars represent 1 standard deviation (s.d.).
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Supplementary Figure S2 1.2
[mRFP1] (relative)
1.0 0.8 0 µM IAM
0.6
50 µM IAM 200 µM IAM
0.4 0.2 0.0 iSAT reaction
70S TX-TL reaction
Supplementary Figure S2. Comparison of iSAT and 70S TX-TL reactions under oxidizing conditions. iSAT and 70S TX-TL reactions were performed with extract that had been pretreated with 0, 50, or 200 µM iodoacetamide (IAM) (final reaction concentration) for 30 min at room temperature. Previously Swartz and colleagues have shown that this treatment results in an oxidizing cell-free protein synthesis reaction environment (Kim and Swartz (2004) Biotechnol. Bioeng., 85, 122-129 and Yin and Swartz (2004) Biotechnol. Bioeng., 86, 188-195). Reactions including the reporter plasmid pY71mRFP1 were incubated at 37°C for 18 h, and fluorescence of translated mRFP1 was measured. Values represent average protein synthesis (n=3) relative to protein synthesis of iSAT or 70S TX-TL reactions with 0 µM IAM treatment and error bars represent 1 s.d.
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Supplementary Figure S3
A254 (relative signal)
0.4
2% PEG 6000 / 2 mM DTBA
0.3
50S 30S
0.2
70S
0.1
0.0 5
6
7
8
9 10 11 12 13 Gradient volume (mL, from top)
14
15
16
17
23S rRNA 16S rRNA
Supplementary Figure S3. Gel electrophoresis analysis of a non-translating iSAT ribosome profile. A 50 µL iSAT reaction with 2% w/v PEG 6000 and 2 mM DTBA and lacking a reporter plasmid was analyzed by ribosome profiling in a 10-40% sucrose gradient. The resulting 375 µL fractions were run by electrophoresis on a 1% agarose gel. The rRNA contained within each fraction was used to identify the predominate peaks as containing 30S or 50S subunits or 70S ribosomes. The peak identities are assumed to hold for all non-translating iSAT ribosome profiles, as peaks appear at the same elution volumes in all traces (see Figure 5A-D of the manuscript).
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Supplementary Figure S4 1.0
2% PEG 6000 / 2 mM DTBA
70S A254 (relative signal)
0.8
50S
0.6
0.4
30S Polysomes
0.2
0.0 5
6
7
8
9
10 11 12 13 Gradient volume (mL, from top)
14
15
16
17
23S rRNA 16S rRNA mRNA
Supplementary Figure S4. Gel electrophoresis analysis of a translating iSAT ribosome profile. A 200 µL iSAT reaction with 2% w/v PEG 6000 and 2 mM DTBA and the reporter plasmid pY71mRFP1-SpA was analyzed by ribosome profiling in a 10-40% sucrose gradient. The resulting 375 µL fractions were run by electrophoresis on a 1% agarose gel. The rRNA contained within each fraction was used to identify the predominate peaks as containing 30S or 50S subunits, 70S ribosomes, or polysomes of multiple 70S ribosomes. The peak identities are assumed to hold for all translating iSAT ribosome profiles, as peaks appear at the same elution volumes in all traces (see Figure 5E-H of the manuscript).
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Supplementary Figure S5
A
B
1.4
iSAT-1
iSAT-2
iSAT-3
1.2
A254
1.0 0.8 0.6 0.4 0.2 0.0 5
C
6
7
0.8
8
70S-1
0.7
9 10 11 12 13 14 15 16 17 Elution volume (mL) 70S-2
70S-3
0.6
A254
0.5 0.4 0.3 0.2 0.1 0.0 5
6
7
8
9 10 11 12 13 14 15 16 17 Elution volume (mL)
Supplementary Figure S5. Data used to determine translation elongation rates of iSAT ribosomes and purified E. coli 70S ribosomes. (A) Average sfGFP production of iSAT and 70S TX-TL reactions with 6% w/v Ficoll 400 and 2 mM DTBA. Values represent average protein synthesis (n=3) with background activity subtracted, and error bars represent 1 s.d. (B, C) Ribosome profiles of (B) iSAT and (C) 70S TX-TL reactions used for translation elongation rate calculations. iSAT reactions were incubated for 2 h, and 200 µL of pooled reactions were loaded onto 10-40% sucrose gradients. 70S TX-TL reactions were incubated for 1 h, and 50 µL of pooled reactions were loaded into 10-40% sucrose gradients.
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Supplementary Figure S6
A
B
Supplementary Figure S6. Comparison of ribosome activity in TX-TL reactions. TX-TL reactions were prepared using ribosome-free S150 extract and reporter plasmid pY71sfGFP combined with either purified iSAT 70S ribosomes, purified native E. coli 70S ribosomes, or commercial E. coli ribosomes from New England Biolabs Inc.(NEB) (P0763S). TX-TL reactions without ribosomes were also prepared to assess residual translation activity of the S150 extract. All reactions included 6% w/v Ficoll 400 and 2 mM DTBA. Reactions were incubated at 37°C and fluorescence of translated sfGFP was measured at (A) 1 h or (B) 18 h. Values represent averages (n≥3) and error bars represent 1 s.d.
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Supplementary Figure S7
A Average ratio of iSAT to E. coli r-proteins
1.5
1.0
0.5
L9 L1 0 L1 1 L1 3 L1 4 L1 5 L1 6 L1 7 L1 8 L1 9 L2 0 L2 1 L2 2 L2 3 L2 4 L2 5 L2 7 L2 8 L2 9 L3 0 L3 1 L3 2 L3 3 L3 4 L3 5 L3 6
L5 L7 L6 /L 12
L4
L3
L2
L1
0.0 50S r-proteins
B Average ratio of iSAT to E. coli r-proteins
1.5
1.0
0.5
S9 S1 0 S1 1 S1 2 S1 3 S1 4 S1 5 S1 6 S1 7 S1 8 S1 9 S2 0 S2 1
S8
S7
S6
S5
S4
S3
S2
S1
0.0 30S r-proteins
Supplementary Figure S7. Proteomic analysis of relative r-protein ratios between purified iSAT 70S ribosomes and purified native E. coli 70S ribosomes for the (A) 50S and (B) 30S subunits. Ribosomes were pelleted by ultracentrifugation through a sucrose cushion, resuspended, and submitted for proteomic analysis. The data indicate that S1, L33, and L36, are underrepresented in iSAT-assembled ribosomes. Values represent averages of 3 replicate readings for 2 independent pairs of samples and error bars represent s.d.
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Supplementary Table S1. DNA sequences of gBlocks used for constructing pY71mRFP1 and pY71mRFP1-SpA. Lowercase letters represent sequence obtained from literature (Chizzolini et al. (2014). ACS Synthetic Biology, 3, 363-371.). Uppercase letters represent additions to the original sequences. Underlined letters indicate cut sites used for construction: NdeI (CATATG), AflII (CTTAAG), or SalI (GTCGAC). Note that a double underline was used for 3’ end of mRFP1, where AflII and SalI overlap (first ‘G’ of double-underlined region).
gBlock name
gBlock sequence
mRFP1
GGTGGTCATatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaa ggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtaccc agaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagt tccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcc cggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggact cctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgc tctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaa accacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggaca tcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccacc ggtgcttaaGTCGACACCACC
Spinach aptamer (SpA)
GGTGGTCTTAAgcccggatagctcagtcggtagagcagcggccggacgcaactgaatgaaatg gtgaaggacgggtccaggtgtggctgcttcggcagtgcagcttgttgagtagagtgtgagctccgtaa ctagtcgcgtccggccgcgggtccagggttcaagtccctgttcgggcgccaGTCGACACCACC
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Supplementary Table S2. Final concentrations of components used in iSAT and 70S TXTL reactions, other than additives described in the text. Note that salt, polyamine, and HEPES-KOH concentrations are total reaction concentrations that include component buffers of S150 extract, r-proteins (TP70), and T7 RNA polymerase. Reagents
Operon-based iSAT reactions
70S TX-TL Reaction
Salts and polyamines: Magnesium glutamate (Mg(Glu)2)
14
mM
14
mM
Ammonium glutamate (NH4(Glu))
10
mM
10
mM
Potassium glutamate (KGlu)
265
mM
265
mM
Spermidine
2.0
mM
2.0
mM
Putrescine
1.5
mM
1.5
mM
ATP
1.20
mM
1.20
mM
GTP
0.85
mM
0.85
mM
UTP
0.85
mM
0.85
mM
CTP
0.85
mM
0.85
mM
Folinic acid
34.0
µg/mL
34.0
µg/mL
tRNA
171
µg/mL
171
µg/mL
E. coli S150 crude extract (MRE600)
3.7
µg/µL
3.7
µg/µL
Operon plasmid: pT7AM552A
4.0
nM
-
Reporter plasmid: pY71mRFP1, pY71mRFP1-SpA, pY71sfGFP, or pK7Luc
4.0
nM
4.0
nM
T7 RNA polymerase
36
ng/µL
36
ng/µL
Transcriptional master mix, consisting of:
Transcriptional and translational components:
Purified 70S ribosomes
-
nM
300
nM
300
nM
-
nM
20 amino acids
2.00
mM
2.00
mM
NAD
0.33
mM
0.33
mM
CoA
0.27
mM
0.27
mM
80.00
mM
80.00
mM
4.00
mM
4.00
mM
42.00
mM
42.00
mM
Total protein of 70S ribosomes (TP70) Other components:
HEPES-KOH, pH 7.6 (total in reaction) Oxalic acid PEP
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Supplementary Table S3. Comparison of translation elongation rates of iSAT ribosomes and 70S E. coli ribosomes with previously published values. Elongation rates for iSAT ribosomes and 70S E. coli ribosomes were determined from translation rate and ribosome profile data, in triplicate, for iSAT reactions after 2 h incubations and 70S TX-TL reactions after 1 h incubations (Supplementary Figure S5) and compared against previously reported values.
iSAT reaction, 2 h 70S TX-TL reaction, 1 h S30 reaction, 30 min* In vivo, exponential growth *
Translation rate (AA/sec) 0.9 ± 0.1 1.6 ± 0.1 1.5 ± 0.2 18
*Underwood, Swartz, and Puglisi (2005) Biotechnol. Bioeng., 91, 425-435
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