RIBOSOMAL PROTECTION FROM TETRACYCLINE MEDIATED BY TET(O) - TET(O) INTERACTION WITH RIBOSOMES IS GTP-DEPENDENT

Tet(O) mediates tetracycline resistance by protecting the ribosome fro m inhibition. A recombinant Tet(O) protein with a histidine tag was pu rified and its activity in protein synthesis characterized. Tetracycli ne inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and process ivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of t he ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the pre sence of the factor were also able to interfere with tetracycline bind ing. Ribosomes increased the affinity of Tet(O)-His for GTP gamma S. T et(O), 70S ribosomes and GTP gamma S formed a complex that could be is olated by gel filtration. The GTP conformer is the active form of Tet( O) that interacts with the ribosome. GTP binding is necessary for Tet( O) activity.