Molecular characterization of the prokaryotic efp gene product involved in a peptidyltransferase reaction.
The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced. We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we report the purification, crystallization and initial characterization of the efp gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin. This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.
Aoki
H
H
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Adams
S L
SL
Turner
M A
MA
Ganoza
M C
MC
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli; D005822:Genetic Vectors; D010452:Peptide Biosynthesis; D010458:Peptidyl Transferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D016335:Zinc Fingers
D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D014157:Transcription Factors; D010458:Peptidyl Transferases