Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly.
Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.
Yamami
Tohru
T
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Ito
Koichi
K
Fujiwara
Toshinobu
T
Nakamura
Yoshikazu
Y
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020043:Genes, Essential; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020653:Peptide Elongation Factor G / Q000235:genetics / Q000502:physiology; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D012269:Ribosomal Proteins / Q000235:genetics / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment
D020653:Peptide Elongation Factor G; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C087267:ribosome releasing factor