3.2. Toe-Printing of Antibiotic-Stalled Ribosomes

The mRNA-ribosome complex can be analyzed by means of reverse transcription with an oligonucleotide annealed downstream to a bound ribosome; the stopping of reverse transcriptase indicates the location of the ribosome on the mRNA. Various translation antibiotics inhibit specific functions of ribosomes, and the site of an arrested ribosome is dependent on the mechanism of the antibiotic’s action. A toe-printing assay can be done directly in a translation reaction in a cell-free transcriptional/translational system composed of purified components. A large set of translation inhibitors affecting the 50S and 30S subunits were tested, and specific patterns of reverse transcription stops were detected [22]. Some antibiotics, such as thiostrepton, clindamycin, tiamulin and pristinamycin IIB, arrest a ribosome on a start codon and could not be distinguished by this method. All of these antibiotics were acting on a large subunit. Another antibiotic group acting on a small subunit, such as streptomycin, kasugamycin and edeine, could not be distinguished because it suppresses all stops of reverse transcriptase. Fortunately, chloramphenicol, florfenicol, linezolid, erythromycin, telithromycin, tylosin, evernimicin, pristinamycin IB, spectinomycin, kanamycin, tetracycline, pactamycin and gentamicin produce a unique, distinguishable pattern of arrested ribosomes. It was shown that this approach is useful for discrimination between known antibiotics while screening culture broths of natural antibiotic producers. Application of this system allows a researcher to avoid the purification and chemical identification of known antibiotics from newly identified producer strains.