Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits. Implications for ribosomal assembly.

Domain I of 23 S RNA of Escherichia coli was probed in renatured RNA, in the protein L24-RNA complex and in 50 S subunits with ribonucleases specific for single- and double-stranded regions and with chemical reagents specific for guanosines (N-1 and N-2), adenosines (N-1, N-7 and N-6), cytidines (N-3) and uridines (N-3). Reactive sites were detected by a reverse transcriptase procedure. The results support most new features of the latest version of the Santa Cruz/Urbana model of the secondary structure, which is based on evidence from sequence comparison. Most double-helical segments were reactive to cobra venom ribonuclease to some degree; the exceptions were the five "long-range" helices that are probably compactly folded within the structure. The data provide evidence for the occurrence of A(syn) X G(anti) pairings in internal loops and at the ends of some helices; they also support the existence of extensive higher-order structuring, especially within the interhelical regions, and are compatible with two of three tertiary interactions in the free RNA that were predicted from comparative sequence studies. Protein L24 is the only primary binding protein that associates with domain I and it strongly protects two sites against ribonuclease and chemical activity. Site A has the properties of a classic protein binding site and we conclude from four lines of evidence that it is the primary attachment site. Site B is rich in highly conserved, unpaired adenosine residues and lies in a potentially critical region of the structure adjoining a group of long-range helices; we infer that L24 binding here is related to the important role of L24 in initiating ribosomal assembly; the existence of both sites is supported, independently, by genetic experiments. L24-induced enhanced reactivities were detected throughout the domain and are consistent with a general "tuning" of the RNA structure. The RNA domain in the 50 S subunits is almost completely resistant to ribonucleases and only a few sites, mainly interhelical, are accessible to chemical reagents. The appearance of several newly reactive nucleotides in the subunit RNA and the enhancement of some others suggest that some minor conformational changes occur on assembly. Nevertheless, the minimal secondary structure of the renatured RNA appears to be retained. We draw the general conclusion that domain I is a highly structured domain that is important for initiating assembly and for the subsequent organization of the ribosome.