Affinity purification of ribosomes with a lethal G2655C mutation in 23 S rRNA that affects the translocation.
A method for preparation of Escherichia coli ribosomes carrying lethal mutations in 23 S rRNA was developed. The method is based on the site-directed incorporation of a streptavidin binding tag into functionally neutral sites of the 23 S rRNA and subsequent affinity chromatography. It was tested with ribosomes mutated at the 23 S rRNA position 2655 (the elongation factor (EF)-G binding site). Ribosomes carrying the lethal G2655C mutation were purified and studied in vitro. It was found in particular that this mutation confers strong inhibition of the translocation process but only moderately affects GTPase activity and binding of EF-G.
Department of Chemistry, Moscow State University, 119899 Moscow, Russia.
Andrei A
AA
Leonov
Petr V
PV
Sergiev
Alexey A
AA
Bogdanov
Richard
R
Brimacombe
Olga A
OA
Dontsova
D001483:Base Sequence; D002455:Cell Division; D018388:Codon, Terminator; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006868:Hydrolysis; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011691:Puromycin / Q000494:pharmacology; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D018388:Codon, Terminator; D011500:Protein Synthesis Inhibitors; D012338:RNA, Ribosomal, 23S; D011691:Puromycin; D019809:Streptavidin; D020558:GTP Phosphohydrolases