Higher-order structure of domain III in Escherichia coli 16S ribosomal RNA, 30S subunit and 70S ribosome.

We have investigated in detail the conformation of domain III of 16S rRNA (nucleotides 913-1408), using a variety of chemical and enzymatic structure probes. The sites of reaction were identified by primer extension with reverse transcriptase using appropriate oligodeoxyribonucleotide primers. This study has been done on 16S rRNA in its naked form, in the 30S subunit and in the 70S ribosome. Data obtained with naked RNA broadly confirm the secondary structure model proposed essentially by comparative sequence analysis, and allow identification of nucleotides involved in tertiary interactions. Our results are in reasonably good agreement with structure probing experiments of Moazed et al. [1]. However, several discrepancies have been observed. Within the 30S subunit, a high number of nucleotides become unreactive whereas other nucleotides show an enhanced reactivity. This probably reflects local conformational changes. Interestingly, they are located in strategic regions of the RNA, e.g. around C1400 (involved in tRNA binding) and C1192 (involved in spectinomycin recognition). Results are also discussed together with the topographical localization of the ribosomal proteins in this area. The study on the 70S particle allows identification of regions at the interface of subunits or exposed at the surface of the ribosome.