In vitro translation assays. Salt-w

In vitro translation assays. Salt-washed ribosomes and separated subunits were
found to perform similarly (Supplementary Fig. 12), and therefore salt-washed
ribosomes were used for all in vitro translation assays unless otherwise noted. In
vitro transcribed mRNAs were translated using the PURExpress D Ribosome Kit
(New England Biolabs). For each reaction, 3 ml of a 4-mM mRNA solution (4 mM
CAT, 4 mM Tte mRNA or a mixture of 0.8 mM CAT and 3.2 mM Tte mRNA) was
re-folded in the presence of 0, 16 or 100 mM preQ1 by heating to 70 C for 2 min,
followed by slow cooling to room temperature for 20 min, and then placed on ice.
The remaining components required for translation were master-mixed and aliquoted
to each reaction (1.5–9 mCi L-[35S]-Cysteine, 4 ml PURExpress Solution A,
1.2 ml Factor Mix and 6 pmol salt-washed ribosomes or separated 30S and 50S
subunits), along with additional preQ1 required to maintain a final concentration
of 0, 16 and 100 mM preQ1 in the final reaction volume of 12 ml. Reactions were
incubated at 37 C for 2 h, frozen on dry ice and stored at 20 C. The following
day, reactions were thawed at room temperature and 2 ml of 1 M KOH was added
to quench the reaction and cleave any remaining peptide from their tRNA. Protein
products were precipitated by adding 5 volumes of cold acetone and pelleted by
centrifugation at 14,000g for 10 min. Pellets were resuspended in 20 ml of 1
loading buffer (45 mM Tris-HCl (pH 8.45 at 25 C), 10% (v/v) glycerol, 50 mM
DTT, 1% (w/v) SDS and 0.01% (w/v) bromophenol blue) and heated at 37 C for
45 min. Protein products were resolved on 16% Tris-tricine SDS–PAGE gels55
electrophoresed at 150 V for 2.5 h. Gels were then fixed for 45 min in 5% (v/v)
glycerol, 40% (v/v) methanol and 10% (v/v) acetic acid and dried on a 3-mm
Whatman paper using a Bio-Rad Model 583 Gel Dryer. Dried gels were imaged
ARTIC using a storage phosphorscreen and Typhoon 9410 Variable Mode Imager
(GE Healthcare Life Sciences). Gel images were quantified using ImageQuant v5.2
(Molecular Dynamics). Unless otherwise noted, after background correction the
intensities for CAT, TTE1564 and TTE1563 bands were divided by their respective
number of cysteine residues (5, 1 and 1, respectively). For each lane, the intensities
for TTE1564 and TTE1563 bands were summed and then divided by the intensity
of the respective CAT band. Values at each preQ1 concentration were graphed in
Prism (GraphPad Software) and an unpaired, two-tailed t-test was used to assess
statistical significance.

Hasil (Bahasa Indonesia) 1:

Tes in vitro terjemahan. Garam-dicuci ribosom dan subunit terpisahditemukan untuk melakukan yang sama (tambahan rajah 12), dan karena itu dicuci garamribosom digunakan untuk semua terjemahan secara in vitro tes kecuali dinyatakan. Dalamvitro ditranskripsi mRNAs telah diterjemahkan menggunakan PURExpress D ribosom Kit(New England Biolabs). Untuk setiap reaksi, 3 ml larutan mRNA 4-mM (4 mMCAT, 4 mM Tte mRNA atau campuran 0.8 mM CAT dan 3.2 mM Tte mRNA)kembali dilipat hadapan 0, 16 atau 100 mM preQ1 oleh pemanasan sampai 70 C selama 2 menit,diikuti oleh lambat pendinginan ke suhu ruang untuk 20 menit, dan kemudian ditempatkan di atas es.Komponen yang tersisa yang diperlukan untuk terjemahan yang dicampur master dan aliquotedreaksi masing-masing (mCi 1.5 – 9 L-[35S]-Sistein, 4 ml larutan PURExpress A,1.2 ml faktor campuran dan 6 pmol garam-dicuci ribosom atau terpisah 30-an dan 50-ansubunit), bersama dengan preQ1 tambahan yang diperlukan untuk mempertahankan kadar akhir0, 16 dan 100 mM preQ1 dalam volume akhir reaksi dari 12 ml. reaksi yangburung di 37 C selama 2 h, beku pada es kering dan disimpan di 20 C. Berikuthari, reaksi yang dicairkan pada suhu kamar dan 2 ml 1 M KOH ditambahkanuntuk memadamkan reaksi dan membelah peptida sisa apapun dari tRNA mereka. Proteinproduk yang dipicu oleh menambahkan 5 volume dingin aseton dan pelleted olehsentrifugasi 14.000 g untuk 10menit pelet itu resuspended dalam 20 ml 1loading penyangga (45 mM Tris-HCl (pH 8.45 25 c), gliserol 10% (v/v), 50 mMDTT, 1% (w/v) SDS and 0.01% (w/v) bromophenol blue) and heated at 37 C for45 min. Protein products were resolved on 16% Tris-tricine SDS–PAGE gels55electrophoresed at 150 V for 2.5 h. Gels were then fixed for 45 min in 5% (v/v)glycerol, 40% (v/v) methanol and 10% (v/v) acetic acid and dried on a 3-mmWhatman paper using a Bio-Rad Model 583 Gel Dryer. Dried gels were imagedARTIC using a storage phosphorscreen and Typhoon 9410 Variable Mode Imager(GE Healthcare Life Sciences). Gel images were quantified using ImageQuant v5.2(Molecular Dynamics). Unless otherwise noted, after background correction theintensities for CAT, TTE1564 and TTE1563 bands were divided by their respectivenumber of cysteine residues (5, 1 and 1, respectively). For each lane, the intensitiesfor TTE1564 and TTE1563 bands were summed and then divided by the intensityof the respective CAT band. Values at each preQ1 concentration were graphed inPrism (GraphPad Software) and an unpaired, two-tailed t-test was used to assessstatistical significance.