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Construction of the ‘minimal’ SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli
Author(s) -
Avdeeva Olga N,
Myasnikov Alexander G,
Sergiev Petr V,
Bogdanov Alexey A,
Brimacombe Richard,
Dontsova Olga A
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02332-3
Subject(s) - signal recognition particle , ribosome , signal recognition particle receptor , translation (biology) , rna , escherichia coli , biology , ribosomal rna , signal peptide , microbiology and biotechnology , signal recognition particle rna , internal ribosome entry site , biochemistry , chemistry , messenger rna , recombinant dna , gene
Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP–ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP‐mediated translation arrest did occur in bacterial cells.