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note
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Phe). The beads were pre-washed three times with buffer containing 0.3% bovine serum albumin (BSA) and once with buffer lacking BSA, and the unbound subunits were removed by washing three times with the 20 μl of binding buffer after the beads were trapped with a magnet.
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Phe in 20 μl of selection buffer. For selection in the presence of paromomycin, 100 μM drug was added. The active subunits were captured with beads and released by addition of 20 μl of 5 mM EDTA, 0.5% SDS, and 300 mM sodium acetate (pH 5.4) and incubation at room temperature for 20 min. Modifications were detected by primer extension with 12 independent primers to scan the entire sequence of 16S rRNA. Modification levels were quantified with a Phosphorimager (Molecular Dynamics), normalized to account for differences in lane loadings, and background levels estimated from unmodified sample lanes were subtracted. Modification of the same set of nucteotides as observed in (9) interfered with P-site tRNA binding, except that the interference at G1401 (N7) was not observed.
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24
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0345286087
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note
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L promoter and cl857, a thermolabile allele of the lambda repressor. Strain DH1 was used as the recipient of the final constructs.
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0344423098
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note
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Phe in 10 μl of selection buffer. The ratio between mutant and wild-type ribosomal subunit population was analyzed by primer extension from allele-specific priming site V as described (16). The mutant population in each mixture was A1492G, 40 ±3%; A1492C, 40 ± 2%; A1493G, 32 ± 2%; A1493C, 23 ± 3%; A1492G-A1493G, 32 ± 3%: and A1492C-A1493C, 43 ± 2%.
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0345286076
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639 → Phe mutant T7 RNA polymerase (29) with 2′ fluoro uridine triphosphate (UTP) (Amersham) or 2′ deoxy-UTP (Sigma) as substrates.
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0344423084
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639 → Phe mutant T7 RNA polymerase gene. Supported by grants from NIH (GMS1266), the Packard Foundation, and the Lucille P. Markey Charitable Trust. S.Y. was supported by a grant from the Japan Society for the Promotion of Science. D.F. was supported by a grant From Institut National de la Santé et de la Recherche Médicale.
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