Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor.
Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced. The P. aeruginosa frr was mapped at 30 to 32 min of the P. aeruginosa chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of E. coli RRF. In an assay including E. coli polysome and elongation factor G, purified recombinant RRF of P. aeruginosa released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr. The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E. coli and Bacillus subtilis. Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.
Ohnishi
M
M
Department of Bacteriology, Shinshu University School of Medicine, Matsumoto, Nagano-Ken 390-8621, Japan. ohnishi7@sch.md.shinshu-u.ac.jp
Janosi
L
L
Shuda
M
M
Matsumoto
H
H
Hayashi
T
T
Terawaki
Y
Y
Kaji
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012269:Ribosomal Proteins; D012270:Ribosomes; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity