title: The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.
abstract: We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.
mesh_terms: D001483:Base Sequence; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014764:Viral Proteins
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; C027729:helix-destabilizing proteins; C036004:ribosomal protein S15; D004265:DNA Helicases