Figure 2.

Chloramphenicol-dependent, UV-induced modifications in E.coli 23S rRNA. (A and B) Ribosomes (0.15 µM) were incubated with (+) and without (–) chloramphenicol (1.2 mM) for 20 min at 37°C, and with (+) and without (–) UV irradiation at 365 nm for 30 min. Ribosomal RNA was isolated and analyzed by primer extension with primers Ec2654 (A) and Ec770 (B). (C) RNase H analysis of the modification at 747. Ribosomes (0.5 µM) were incubated with (+) and without (–) chloramphenicol (1.2 mM) for 20 min at 37°C, irradiated at 365 nm for 30 min, followed by isolation of rRNA. A small portion of each sample (70S) was analyzed directly by extension from primer Ec770. The remainder of each sample was treated with RNase H in the presence of oligonucleotides Ec617 and Ec820. The released fragment (Frag.) was gel-purified and subjected to primer extension using primer Ec770. In (B) and (C), 23S rRNA from the E.coli IB10 strain lacking the m¹G745 methyltransferase and the strong stop at G745 was used for the sequencing tracks (43). The rRNA modification-induced stop is displaced by one base below the corresponding stop in the sequencing tracks (G, A, U and C).