Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2005
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
Supporting Information
for
Termination-Free Prokaryotic Protein Translation Using
Anticodon-Adjusted E. coli tRNASer as Unified Suppressors of the
UAA/UGA/UAG Stop Codons. Read-Through Ribosome-Display of
Full-Length DHFR with Translated UTR as a Buried Spacer Arm
Atsushi Ogawa, Shinsuke Sando, and Yasuhiro Aoyama
General: Reagents and solvents were purchased from standard suppliers and used
without further purification. All enzymes used in this study were from commercial
sources. Two types of reconstituted prokaryotic cell-free translation systems (PURESYSTEM classic I) RFs(+) and RFs(∆) containing plenty and diminished amounts of
release factors (PURESYSTEM custom), respectively, were purchased from Post
Genome Institute Co., Ltd. Details are described in the reference.[7]
Preparation of Template DNAs: pDHFR, encoding E. coli DHFR, was a gift from Dr. Y.
Shimizu of Post Genome Institute Co., Ltd. All templates were prepared by successive PCRs carried out in 20 µL of a reaction mixture containing 4 pmol of forward primer, 4 pmol of reverse primer, 20 ng of pDHFR for the first PCR or ~20 fmol of purified
(agarose gel electrophoresis) PCR product for the subsequent PCRs, 1.25 U of Pfu
Ultra HF DNA polymerase (Stratagene), 4 nmol each of dNTPs (TOYOBO), and 2 µL
of 10× Pfu Ultra HF reaction buffer. The sequences of the forward and reverse primers
used are summarized below.
In Vitro Transcription of Template DNAs: Template mRNAs were obtained by run-off
transcription of the DNA templates prepared as above using a T7-MEGAshortscript Kit
(Ambion). Briefly, a T7-transcription mixture (10 µL) containing 3 µL of the PCR solution of template DNA was incubated at 37 °C for 2 h. To the reaction mixture was added 1 U of DNase I and the mixture was incubated for additional 15 min. The transcribed mRNAs were purified with an RNeasy MinElute Cleanup Kit (QIAGEN). Concentrations of the purified mRNAs were determined by the absorbance at 260 nm.
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Formation of PRM Complexes and T7-tag Based Selection: Read-through translation
of a stop codon-containing mRNA template (2 µg) was carried out in 25 µL of a reconstituted RFs(∆) translation system in the presence of suppressor tRNA (5 µg) at 37 °C
for 10 min. Read-through irrelevant translation using a stop-codon-free template or in
the absence of suppressor tRNA was carried out under either RFs(+) or RFs(∆) conditions. To the solution was added 455 µL of ice-cold selection buffer (Phosphate-K, pH
7.3) containing 92.2 mM of K+, 300 mM of Na +, 50 mM of Mg2+, Tween20 (WAKO)
(0.05%), and Block Ace (Dainippon Pharmaceutical) (2%), followed by addition of 20
µL of T7-tag antibody agarose (Novagen). The mixture was gently inverted for 30 min
at 4 °C, transferred to an empty filter-column (Microspin Columns, Amersham), and
filtered by centrifugation. The agarose remaining on the filter was washed five -times
with 200 µL of ice-cold selection buffer (total 1,000 µL) and finally resuspended in 200
µL of elution buffer (Phosphate-K, pH 7.2) containing 92.2 mM of K+, 300 mM of Na +,
30 mM of EDTA, Tween20 (0.05%), and Block Ace (2%). The resulting suspension
was gently inverted for 10 min at room temperature and mRNAs, released from the
PRM complex, were eluted by centrifugation. The mRNAs recovered were purified
with an RNeasy MinElute Cleanup Kit and the recovery yields were determined by
measuring the UV absorbance at 260 nm. The recovered mRNAs were also RT-PCR
amplified using a QIAGEN One-Step RT-PCR kit (QIAGEN) with a forward primer
5'-GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC CTC TAG
AAA TAA TTT TGT TTA ACT TTA AGA AGG AGA TAT ACC A-3’ and a reverse primer
5'-CCG CCG CTC CAG AAT CTC-3' or 5’-CAA AGG TAC GCC GGA AGG TAT ATA
CG-3’ in case of T7(+)/T7(-) competition experiments.
Preparation of suppressor tRNASerUXYZ : E. coli suppressor tRNA SerUXYZ (XYZ = UUA,
UCA, or CUA) were prepared by run-off in vitro transcription. Template DNAs were
prepared by PCR in 20 µL of a reaction mixture containing 20 pmol of forward primer
5’-G TAA TAC GAC TCA CTA TA GGA GAG ATG CCG GAG CGG CTG AAC-3’, 20
pmol of reverse primer 5’-TGG CGG AGA GAG GGG GAT TTG AAC CCC CGG TAG
AGT TGC C-3’, 100 fmol of template DNA 5’-GGA GAG ATG CCG GAG CGG CTG
AAC GGA CCG GTC TXY ZAA ACC GGA GTA GGG GCA ACT CTA CCG GGG GTT
CAA ATC CCC CTC TCT CCG CCA-3’, 1.25 U of Pfu Ultra HF DNA polymerase (Stratagene), 10 nmol each of dNTPs (TOYOBO), and 2 µL of 10× Pfu Ultra HF reaction
buffer. Transcription was carried out in 500 µL of a reaction mixture containing 2500 U
of T7 RNA polymerase (TOYOBO), 2 µmol each of NTPs (Fermentas), 5 µmol of GMP
(Sigma), 1U of inorganic pyrophosphatase (Calbiochem-Novabiochem), 80 U of
RNase inhibitor (TOYOBO), 18 µL of PCR solution of template DNA, and 50 µL of 10×
reaction buffer (37 °C for 18 h). The transcribed tRNA was purified by denaturing
PAGE (8%), followed by ethanol precipitation, and then redissolved in 500 µL of water.
The tRNA obtained was further purified using Microcon YM-30 (Millipore) and G-25
Microspin Columns (Amersham).
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Competition of T7(+) and T7(-) templates:
Figure S1. Selective recovery of the T7(+)Stop(+) template using a 1:1 mixture of
T7(+)Stop(+) and T7(-)Stop(-) templates under competitive conditions in a reconstituted
translation system.
PCR Primers for Preparation of Template DNAs:
(T7 promoter and T7-tag sequences are underlined and italicized, respectively.)
T7(+)Stop(-) templates
First PCR
Forward primer: 5'-AAGGAGATATACCAATGATCAGTCTGATTG-3'
Reverse primer: 5'-CCGCCGCTCCAGAATCTC-3'
Second PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3'
Reverse primer: 5'-GCATCCGGCGCTAGCCGTAAATTCTATACAAAAACCCATTTGCTGTCCACCAGTCATGCTAGCCATCCGCCGCTCCAGAATCTCAAAGC-3'
Third PCR to generate T7(+)Stop(-)(78)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-AACACAGCCTGATATAGGAAGGCCGGATAAGACGCGACCGGCGTCGCATCCGGCGCTAGCCGTAAATTC-3'
Forth PCR to generate T7(+)Stop(-)(150)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CTGGGAAGATAAACAGTATTTTGTCCAGCCGTCGAACCGGCATAAGGATTTGGGCGAAGCGGCGGCGTCTAAAACACAGCCTGATATAGGAAG-3'
Fifth PCR to generate T7(+)Stop(-)(222)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CAAAGGTACGCCGGAAGGTATATACGCGCTGGATACCGGCTGCTGCTGGGGTGGTACATTAACCTGCCTGCGCTGGGAAGATAAACAGTATTTTGTC-3'
T7(-)Stop(-) templates
First PCR
Forward primer: 5'-AAGGAGATATACCAATGATCAGTCTGATTG-3'
Reverse primer: 5'-CCGCCGCTCCAGAATCTC-3'
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Second PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3'
Reverse primer: 5'-GCATCCGGCGCTAGCCGTAAATTCTATACAAAACCGCCGCTCCAGAATCTCAAAGC-3'
Third PCR to generate T7(-)Stop(-)(78)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-AACACAGCCTGATATAGGAAGGCCGGATAAGACGCGACCGGCGTCGCATCCGGCGCTAGCCGTAAATTC-3'
Forth PCR to generate T7(-)Stop(-)(150)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CTGGGAAGATAAACAGTATTTTGTCCAGCCGTCGAACCGGCATAAGGATTTGGGCGAAGCGGCGGCGTCTAAAACACAGCCTGATATAGGAAG-3'
Fifth PCR to generate T7(-)Stop(-)(222)
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CAAAGGTACGCCGGAAGGTATATACGCGCTGGATACCGGCTGCTGCTGGGGTGGTACATTAACCTGCCTGCGCTGGGAAGATAAACAGTATTTTGTC-3'
T7(+)UAA(+)UAA(+)(222), T7(+)UGA(+)UGA(+)(222), and
T7(+)UAG(+)UAG(+)(222)
(XYZ = TTA, TCA, or CTA for the UAA, UGA, or UAG template, respectively)
First PCR
Forward primer: 5'-AAGGAGATATACCAATGATCAGTCTGATTG-3'
Reverse primer: 5'-CCGCCGCTCCAGAATCTC-3'
Second PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3'
Reverse primer: 5'-GCATCCGGCGCTAGCCGTAAATTCTATACAAAAXYZACCCATTTGCTGTCCACCAGTCATGCTAGCCAT CCGCCGCTCCAGAATCTCAAAGC-3'
Third PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-AACACAGCCTGATATAGGAAGGCCGGATAAGACGCGACCGGCGTCGCATCCGGCGCTAGCCGTAAATTC-3'
Forth PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CTGGGAAGATAAACAGTATTTTGTCCAGCCGTCGAACCGGCATAAGGATTTGGGCGAAGCGGCGGCGTCXYZAACACAGCCTGATATAGGAAG-3'
Fifth PCR
Forward primer: 5'-GAAATTAATACGACTCACTATAGGGAG-3'
Reverse primer: 5'-CAAAGGTACGCCGGAAGGTATATACGCGCTGGATACCGGCTGCTGCTGGGGTGGTACATTAACCTGCCTGCGCTGGGAAGATA AACAGTATTTTGTC-3'
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