ABSTRACT
Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coli and Thermus thermophilus S7 with a fragment of the 3' domain of the E. coli 16S rRNA. Both proteins showed a high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA-protein interactions.
Subject(s)
RNA, Ribosomal, 16S/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Conformation , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal, 16S/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomes/genetics , Thermus thermophilus/genetics , Thermus thermophilus/metabolismABSTRACT
Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research. To this end, when studying protein S7, we created an E. coli overproducer of the recombinant protein S7 of Thermus thermophilus. The vector for expression was pQE-32 having a strong promoter of E. coli phage T5 and six triplets of His at the 5'-end. This N-terminal six His tag of the fusion protein is responsible for binding to Ni-NTA-resin and allows purifying the protein in one step. The yield of the recombinant protein was 20% and more of the total cellular proteins. In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E. coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52%. This fact provides a base for the system to study functions of individual proteins.