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Biochemistry (Moscow)

, Volume 75, Issue 7, pp 841–850 | Cite as

Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon

  • A. V. Surdina
  • T. I. Rassokhin
  • A. V. Golovin
  • V. A. Spiridonova
  • A. M. KopylovEmail author
Article
  • 52 Downloads

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.

Key words

ribosomal biogenesis S7 protein streptomycin operon bacteria translation regulation regulome SELEX SERW 

Abbreviations

ICR (intercistronic region)

region of E. coli str mRNA between cistrons S12 and S7

r6hEcoS7

recombinant protein S7 of E. coli with six N-terminal His residues

r6hTthS7

recombinant protein S7 of Thermus thermophilus with six N-terminal His residues

r-proteins

ribosomal proteins

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

serwamer

RNA fragment obtained by SERW and capable of protein binding

str

streptomycin

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Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • A. V. Surdina
    • 1
  • T. I. Rassokhin
    • 1
  • A. V. Golovin
    • 2
  • V. A. Spiridonova
    • 1
  • A. M. Kopylov
    • 1
    • 3
    Email author
  1. 1.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Faculty of Bioengineering and BioinformaticsLomonosov Moscow State UniversityMoscowRussia
  3. 3.Chemical FacultyLomonosov Moscow State UniversityMoscowRussia

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