Spontaneous ribosome bypassing in growing cells

Translating ribosomes can pass through a stretch of messenger RNA without translating and resume protein chain elongation after the bypassed region. We previously investigated the stimulation of bypassing when the codon in the ribosomes A-site called for an aminoacyl-tRNA species in short supply. Here, we investigate bypassing in unstarved, growing cells. A collection of lacZ bypass reporters was constructed with nearly all the sense codons as the "takeoff site", each with its matched landing site 16 nucleotides downstream in the β-galactosidase reading frame. β-galactosidase synthesis in unstarved cells carrying these reporters was found to vary over a large range. The takeoff sites UUU and AGG yielded unusually high enzyme activities, sufficient for protein sequence analysis; in these cases, sequencing (by Edman degradation or by mass spectrometry) confirmed that the synthesis of lacZ protein occurred through the 16 nt bypass from takeoff to landing site. Thus, bypassing occurs spontaneously under normal conditions, and is not limited to the pathology of amino acid starvation. Indirect evidence suggests that most of the lower enzyme activities of the rest of the collection also reflects bypassing. Another collection of reporters was made various triplets in the A-site; a UUC the codon immediately following the takeoff triplet. Spontaneous bypassing in representatives of this collection varied roughly inversely with the abundance of the tRNA encoded at the A-site. For two A-site codons tested, introduction of additional copies of the relevant tRNA gene on a second plasmid reduced spontaneous bypassing. We conclude that any pause with the A-site empty stimulates bypassing. From the P-site and A-site effects on bypassing, we estimated the average frequency of ribosome takeoff; from this, we calculate that the probability that a ribosome will succeed in translating the entire lacZ coding sequence is about 0.73, in agreement with earlier, independent estimates.