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32P-end-labeling with polynucleotide kinase.
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4TCPm was observed.
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2642683154
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note
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2 and 330 mM KCl was used. Complexes were exposed for various lengths of time to a hand-held UV light source (366 nm) (UVL-56, Blak-Ray Lamp, UVP) at a distance of 3 cm (27). The intact rRNAs were then extracted with phenol and resolved on 3.8% polyacrylamide gels containing 7 M urea.
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14
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2642616939
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note
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32P-radiolabeled crosslinked 23S rRNA species was digested with RNase T1 in 0.2 M NaOAc (pH 5.5) containing 2.5 mM EDTA at 37°C for 20 min and the products were resolved on 24% polyacrylamide gels containing 6 M urea.
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4TCPm compound using cDNA 2639 (5′-CTAGGAGCAGCCCCCC-3′) (15).
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2 and 33% MeOH] with CACCA-(N-Ac-Phe) (2 μM). Aliquots were removed from the reaction mixture at the indicated times, the rRNAs were extracted with phenol and digested with RNase T1, and the fragments were resolved on 24% polyacrylamide gels containing 6 M urea.
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25
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4TCPm and a single time point (2 min) of exposure to UV light (in the linear range of the reaction) under standard conditions. Antibiotic inhibition of peptidyl transferase activity was performed with the fragment P-site substrate, CACCA-(N-Ac-Phe), supplied at a concentration of 0.3 μM; a single linear time point of 4 min was used for analysis.
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4OAc (pH 5.4) at 37°C for 120 min. Np (Ap, Cp, Gp, and Up) products were resolved by paper electrophoresis at pH 3.5 (pyridine acetate) (30 min at 3000 V). The integrity of the 3′ termini of the mutant tRNAs was at least 75% correct (8).
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We thank B. Cormack, G. Culver, L. Holmberg, K. Fredrick, K. Lieberman, and E. Strauss for helpful comments on the manuscript and S. Joseph and C. Merryman for helpful discussions. Supported by grants from the National Institutes of Health, the National Science Foundation, the Lucille P. Markey Charitable Trust to the Center for Molecular Biology of RNA, and a Burroughs-Wellcome Career Award to R.G.
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