title: Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.
abstract: In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Surdina
authors.forename: T I
authors.initials: TI
authors.lastname: Rassokhin
authors.forename: A V
authors.initials: AV
authors.lastname: Golovin
authors.forename: V A
authors.initials: VA
authors.lastname: Spiridonova
authors.forename: A M
authors.initials: AM
authors.lastname: Kopylov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D013307:Streptomycin / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C037442:ribosomal protein S7; D013307:Streptomycin