The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

摘要: The peptidyl transfer site has been localized at the centre of domain V 23S-like ribosomal RNA (rRNA) primarily on basis a chloramphenicol binding site. implicated region constitutes an unstructured circle in current secondary structural model which contains several universally conserved nucleotides. With view to investigate function this further, four these nucleotides, including one indirectly binding, were selected for mutation Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs expressed vivo plasmid-encoded (rrnB) operon and each yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions inviable it was shown by inserting mutated genes after temperature-inducible promoter that mutant directly responsible. In addition, G2502----A transition caused decreased growth rate, probably due partial selection against ribosome incorporation into polysomes, while A2503----C transversion produced rate conferred resistance chloramphenicol. All incorporated 50S subunits, but two lethal strongly 70S ribosomes, preferred over chromosome-encoded RNA, contrary regulatory theories. results establish critical functional importance highly nucleotides region. A mechanistic is also presented explain disruptive effect (and other antibiotics) peptide bond formation subunit interface.