Mapping the Ribosomal Protein S7 Regulatory Binding Site on mRNA of the E. coli Streptomycin Operon

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A. V. Surdina¹, T. I. Rassokhin¹, A. V. Golovin², V. A. Spiridonova¹, and A. M. Kopylov^1,3*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3181; E-mail: kopylov.alex@gmail.com

²Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

³Chemical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received December 17, 2009; Revision received March 23, 2010

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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.

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KEY WORDS: ribosomal biogenesis, S7 protein, streptomycin operon, bacteria, translation regulation, regulome, SELEX, SERW

DOI: 10.1134/S0006297910070059

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