CONFORMATIONAL-ANALYSIS OF ESCHERICHIA-COLI 30S RIBOSOMES CONTAINING THE SINGLE-BASE MUTATIONS G530U, U1498G, G1401C, AND C1501G AND THE DOUBLE-BASE MUTATION G1401C C1501G/

Biochemical and genetic studies have pointed out the importance of sev eral sites in 16S ribosomal RNA of Escherichia coli in the decoding pr ocess. These sites consist of the core of the decoding center (1490/15 00 region) and two other segments (530 and 1050/1200 regions). To dete ct a possible structural link between these functionally related regio ns, we analyzed their sensitivity to conformational changes induced by mutations which are located in each of these regions and are known to affect the decoding process. The conformations of five segments of 16 S rRNA (1-106, 406-569, 780-978, 997-1247, and 1334-1519) were analyze d by chemical probing of 30S ribosomes containing the following mutati ons: G530U, U1498G, G1401C, C1501G, and G1401C/C1501G. Ribosomes recon stituted with natural wild-type 16S RNA showed only minor conformation al differences with respect to ribosomes isolated from cells. When 16S RNA made in vitro replaced natural 16S RNA, a slightly looser conform ation of the central core region was found. Mutant ribosomes made by r econstitution with mutant 16S RNA made in vitro showed conformational effects which were in all cases localized to the region of secondary s tructure surrounding the site of mutation. Although the core of the de coding center (1400/1500 region) and the two other sites (530 and 1050 /1200 regions) participating in the decoding function have been functi onally linked, our data indicate that they are structurally independen t. They also provide evidence for an unusual structure of the 1400/150 0 decoding center, possibly involving noncanonical interactions. Furth ermore, the absence of any conformational effect induced by the G530U mutation except at the site of mutation itself points to its direct, a s opposed to indirect, involvement in the decoding function of the rib osome.