Mapping the Ribosomal Protein S7 Regulatory Binding Site on mRNA of<i> E. coli</i> Streptomycin Operon
In this research an application of deletion analysis of the intercistronic region (ICR) to study the mRNA binding site for E. coli recombinant protein S7 (r6hEcoS7) revealed that just an 80-nucleotide long fragment is enough. A model of bipartite interaction of S7 with the ICR is suggested. These two mRNA sites are the region of stem bifurcation and SD sequence of the S7 cistron. By selecting a random RNA library based on E. coli ICR, the RNA-binding site for T. thermophilus S7 protein (r6hTthS7) was created de novo. It has a single apparent recognition site only - within the stem bifurcation region. The Selection of Random RNA Window (SERW) method has been applied for two ICR RNA libraries, in which five nucleotides were randomized next to the bifurcation region. First library has authentic AG (-82/-20), and in the other one it has been mutated to AU. The serwamer which binds to r6hTthS7 specifically has been selected, and it turned out to be a mutant RNA68. The serwamer binds to r6hTthS7 with the same affinity as authentic intercistron str-mRNA does to r6hEcoS7 with apparent dissociation constants 89 ± 43 and 50 ± 24 nM, respectively.