Ribosomal targets for antibiotic drug discovery
The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.
1. A method to identify a compound that interferes with ribosomal function which comprises
(a) surface-immobilizing a ribosome having a FRET pair sensitive to transitioning between low FRET and high FRET states under translation competent conditions, wherein said FRET pair comprises a first fluorophore attached to a ribosomal protein of said ribosome and a second fluorophore attached a ribosomal protein of said ribosome;
(b) adding a test compound to said ribosome; and
(c) monitoring or detecting changes in FRET states using smFRET imaging techniques to identify a test compound capable of
(i) stabilizing said ribosome in an intermediate FRET state,
(ii) changing said ribosome's distribution into or out of an intermediate FRET state, or
(iii) changing said ribosome's rate of transition into or out of an intermediate FRET state.
2. The method of claim 1 , wherein said first fluorophore is attached to ribosomal protein L1 and said second fluorophore is attached to ribosomal protein S13.
3. The method of claim 2 , wherein the fluorophore attached to L1 is at (T202C) L1 and the fluorophore attached to S13 is at or near the amino terminus of S13.
4. The method of claim 1 , wherein said compound is identified as a candidate antibiotic when said test compound
(i) stabilizes said ribosome in an intermediate FRET state or in a high FRET state,
(ii) increases said ribosome's distribution in intermediate or high FRET states,
(iii) increases said ribosome's rate of transition into intermediate or high FRET states, or
(iv) abolishes FRET.
5. The method of claim 1 , wherein said intermediate FRET state comprises the P/pe tRNA binding state.
6. The method of claim 1 , wherein said FRET pair consists of a donor-acceptor fluorophore pair or a donor-quencher fluorophore pair.
7. The method of claim 1 , wherein said first fluorophore is attached to a ribosomal protein in a large ribosomal subunit and said second fluorophore is attached to a ribosomal protein in a small ribosomal subunit.