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Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli.

Molecular Microbiology

biosynthesis, genetics, Base Sequence, Binding Sites, Codon, DNA Mutational Analysis, Escherichia coli, metabolism, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Nucleic Acid Conformation, Plasmids, Protein Binding, Bacteriophage lambda, Protein Biosynthesis, RNA, Messenger, chemistry, Recombinant Fusion Proteins, Restriction Mapping, Ribosomal Proteins, Transcription, Genetic, beta-Galactosidase

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      Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

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