In vivo chemical footprinting of the Escherichia coli ribosome

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Abstract
We have studied the in vivo chemical accessibility of 16S rRNA residues A349-G1505 in the small subunit of the Escherichia coli ribosome. Exponentially growing E. coli cultures were reacted with dimethyl sulfate, and the reactive sites on the 16S ribosomal RNA were analyzed by reverse transcription, an assay which detects reactions at N1-A and N3-C. In agreement with previous in vitro results, three regions of 16S RNA appeared particularly reactive to dimethyl sulfate: hairpin 27 (residues A892-A915) of the central domain, and hairpin 33-33A (residues A994-C1037) and the tip of hairpin 41 (residues A1256-A1275), both from the 3' major domain. These three regions contained 52% of the reactive residues but only 8% of the residues scanned. In contrast to previous in vitro results, three small sections of 16S RNA appeared protected: the tip of hairpins 26 (residue A845) and 31 (residues A968-A969), and residues A1418, A1441, and A1483 of the middle body of hairpin 44. Four of the dimethyl sulfate reactive sites (A831, C948, A1019, and C1192) are located in positions usually assumed to be double-stranded (helices 26, 30, 33-33A, and 34), which suggests alternative structures for these helices at least during part of the translation process, as if the residues in question belonged to "conformational switches." The addition of chloramphenicol protected residues A831, A1035-A1036, and A1503, which suggests that they belong to the mobile regions of the elongating ribosome, and become exposed during some transition(s) from one ribosomal state to the other during the elongation cycle.

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