Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon
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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.
Key wordsribosomal biogenesis S7 protein streptomycin operon bacteria translation regulation regulome SELEX SERW
- ICR (intercistronic region)
region of E. coli str mRNA between cistrons S12 and S7
recombinant protein S7 of E. coli with six N-terminal His residues
recombinant protein S7 of Thermus thermophilus with six N-terminal His residues
- SERF (Selection of Random RNA Fragments)
selection of library of given RNA random fragments
- SERW (Selection of Random RNA Windows)
selection of library for basic RNA structure with randomized site several nucleotides in size
RNA fragment obtained by SERW and capable of protein binding
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