| Description |
Translational bypassing joins the information found within two disparate open reading frames into a single polypeptide chain. The mechanism involves three stages: take-off, scanning, and landing. In takeoff, the peptidyl-tRNA-mRNA complex in the P-site of the ribosome dissociates. In scanning, the peptidyl-tRNA probes the mRNA sliding through the P-site. In landing, the peptidyl-tRNA re-pairs with a codon to which it can form a stable interaction. In the best characterized example - 4 gene 60 - a complex set of signals stimulates bypassing of 50 nucleotides (nt) between the two open reading frames. Efficient bypassing requires four signals: matching GGA codons flanking the 50-nt coding gap, a stop codon just downstream of the take-off site GGA, an mRNA stem-loop structure that folds at the junction between the first open reading frame the coding gap, and a cis-acting signal contained in a stretch of amino acids encoded by the first open reading frame. Normally, bypassing competes poorly with decoding in the A-site-background rates of bypassing are generally less than < 1%. In gene 60, bypassing efficiency approaches 50%. The stem-loop and the nascent peptide signal ensure efficient bypassing by preventing recognition of the stop codon by release factors and promoting peptidyl-tRNA slippage. A genetic selection identified mutant tRNA[Gly/2] variants that reduce bypassing efficiency three- to five-fold. The mutations, which either reduce the stability of the elbow region or destabilize the anticodon stem, apparently reduce the ability of tRNA to re-pair at the landing site GGA. Mutations that inactivate the nascent peptide signal restore bypassing, suggesting that the nascent peptide remains active subsequent to take-off. Nearly all ribosomes initiate bypassing at the take-off site GGA, but only half resume translation in the second open reading frame. Factors known to promote premature dissociation of peptidyl-tRNA from the ribosomes do not have a major influence on bypassing efficiency, suggesting that scanning ribosomes may be inherently unstable. A model for bypassing is proposed in which the stem-loop limits recognition of the UAG at the end of the first open reading frame and the nascent peptide promotes dissociation of peptidyl-tRNA-mRNA pairing. |
