Single-molecule correlated chemical probing reveals large-scale structural communication in the ribosome and the mechanism of the antibiotic spectinomycin in living cells

The ribosome moves between distinct structural states and is organized into multiple functional domains. Here, we examined hundreds of occurrences of pairwise through-space communication between nucleotides in the ribosome small subunit RNA using RING-MaP single-molecule correlated chemical probing in live bacterial cells. RING-MaP revealed four structural communities in the small subunit RNA, each distinct from the domain organization defined by the RNA secondary structure. The head domain contains two structural communities: The outer-head contains the pivot for head swiveling, and an inner-head community is structurally integrated with the long helix 44 and spans the entire ribosome intersubunit interface. In-cell binding by the antibiotic spectinomycin barely perturbs its local binding pocket as revealed by the per-nucleotide chemical probing signal. In contrast, spectinomycin stabilizes long-range RNA-RNA contacts that extend roughly 95 Å across the ribosome to connect the pivot for head swiveling with the axis of intersubunit rotation. The two major motions of the small subunit – head swiveling and intersubunit rotation – are thus coordinated via long-range structural communication, which is specifically modulated by spectinomycin. Single-molecule correlated chemical probing revealed a new ribosome domain structure, and rationalizes the profound functional effects of binding by a low-molecular-mass antibiotic to the megadalton ribosome complex.