Evidence that the bypassing ribosome travels through the coding gap.

In translational bypassing, a peptidyl-tRNA::ribosome complex skips over a number of nucleotides in a messenger sequence and resumes protein chain elongation after a "landing site" downstream of the bypassed region. The present experiments demonstrate that the complex "scans" processively through the bypassed region. This conclusion rests on three observations. (i) When two potential "landing sites" are present, the protein sequence of the product shows that virtually all ribosomes land at the first and virtually none at the second. (ii) In such a sequence with two landing sites, the presence of a terminator triplet in phase in the coding region immediately after the first landing site drastically reduces the efficiency of bypassing. (iii) Internally complementary sequences that can form a stable stemloop in the bypassed region significantly reduce the efficiency of bypassing. We analyze bypassing from a given "takeoff" site to "landing sites" at different distances downstream so as to derive estimates of the frequency of ribosome takeoff and of the stability of the bypassing complex.