title: Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding.
abstract: Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921-922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC-SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical Center, MO 63104, USA.
authors.forename: G
authors.initials: G
authors.lastname: Ericson
authors.forename: P
authors.initials: P
authors.lastname: Minchew
authors.forename: P
authors.initials: P
authors.lastname: Wollenzien
mesh_terms: D000447:Aldehydes; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002074:Butanones; D003028:CME-Carbodiimide / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004121:Dimethyl Sulfoxide; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009690:Nucleic Acid Conformation; D011072:Poly U / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000447:Aldehydes; D002074:Butanones; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012360:RNA, Transfer, Phe; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; D011072:Poly U; C005135:kethoxal; D008274:Magnesium; D004121:Dimethyl Sulfoxide