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 several sites in 16S ribosomal RNA of Escherichia coli in the decoding process. These sites consist of the core of the decoding center (1400/1500 region) and two other segments (530 and 1050/1200 regions). To detect a possible structural link between these functionally related regions, 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 16S rRNA (1-106, 406-569, 780-978, 997-1247, and 1334-1519) were analyzed by chemical probing of 30S ribosomes containing the following mutations: G530U, U1498G, G1401C, C1501G, and G1401C/C1501G. Ribosomes reconstituted with natural wild-type 16S RNA showed only minor conformational differences with respect to ribosomes isolated from cells. When 16S RNA made in vitro replaced natural 16S RNA, a slightly looser conformation of the central core region was found. Mutant ribosomes made by reconstitution with mutant 16S RNA made in vitro showed conformational effects which were in all cases localized to the region of secondary structure surrounding the site of mutation. Although the core of the decoding center (1400/1500 region) and the two other sites (530 and 1050/1200 regions) participating in the decoding function have been functionally linked, our data indicate that they are structurally independent. They also provide evidence for an unusual structure of the 1400/1500 decoding center, possibly involving noncanonical interactions. Furthermore, the absence of any conformational effect induced by the G530U mutation except at the site of mutation itself points to its direct, as opposed to indirect, involvement in the decoding function of the ribosome.
Moine
H
H
UPR9002, Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, France. moine@ibmc.u-strasb.fr
Nurse
K
K
Ehresmann
B
B
Ehresmann
C
C
Ofengand
J
J
D001483:Base Sequence; D003597:Cytosine Nucleotides / Q000235:genetics; D003857:Deoxyuridine; D004926:Escherichia coli / Q000235:genetics; D006150:Guanine Nucleotides / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D003597:Cytosine Nucleotides; D006150:Guanine Nucleotides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D003857:Deoxyuridine