Distance-dependent inhibition of translation initiation by downstream out-of-frame AUGs reveals that ribosome scans in a Brownian ratchet process

Abstract

While eukaryotic ribosomes are widely presumed to scan mRNA for the AUG codon to initiate translation in a strictly 5′–3′ movement (strictly unidirectional scanning model), other evidence has suggested that the ribosome uses small-amplitude 5′–3′ and 3′–5′ oscillations with a net 5′–3′ movement to recognize the AUG codon (Brownian ratchet scanning model). Here, we generated 13,437 yeast variants, each with an ATG triplet placed downstream (dATGs) of the annotated ATG (aATG) codon of green fluorescent protein. We found that out-of-frame dATGs could inhibit translation at the aATG, but with diminishing strength over increasing distance between aATG and dATG, undetectable beyond ∼17 nt. Computational simulations revealed that each triplet is scanned back and forth approximately ∼10 times until an AUG codon is recognized. Collectively, our findings uncover the basic process by which eukaryotic ribosomes scan for initiation codons, and how this process could shape eukaryotic genome evolution and influence cancer development.

Translational buffering by ribosome stalling in upstream open reading frames

“…In the idealized case of homogeneously sized ribosomes (30 nt footprints [ 56 , 71 ]) and strict 5′-3′ scanning, d stall must be an integer multiple of 30 nt for buffering to occur. This strong dependence of buffering on d stall is relaxed when backward scanning [ 41 , 72 – 74 ] occurs with a high rate ( S2D Fig , middle). To simplify our modeling interpretations, we considered UL4 uORF2, that is 22 codons long, to be 21 codons so that a queue behind the terminating ribosome stall positions a scanning ribosome’s P-site exactly at the start codon.…”

“…dependence of buffering on d stall is relaxed when backward scanning [41,[72][73][74] occurs with a high rate (S2D Fig, middle). To simplify our modeling interpretations, we considered UL4 uORF2, that is 22 codons long, to be 21 codons so that a queue behind the terminating ribosome stall positions a scanning ribosome's P-site exactly at the start codon.…”

Translational buffering by ribosome stalling in upstream open reading frames

“…In the idealized case of homogeneously sized ribosomes (30 nt footprints 55,68 ) and strict 5′-3′ scanning, 𝑑 𝑠𝑡𝑎𝑙𝑙 must be an integer multiple of 30 nt for buffering to occur. This strong dependence of buffering on 𝑑 𝑠𝑡𝑎𝑙𝑙 is relaxed when backward scanning 41,[69][70][71] occurs with a high rate (Fig. S2D, middle panel).…”

“…5A, right panel, yellow-green line). While there are estimates of how far ribosomes can backward scan [69][70][71] , we are not aware of any backward scanning rate estimates. It is unlikely that the rate of backward scanning approaches the rate of forward scanning (5 nt/s here) given the 5′-3′ directionality of scanning.…”

“…5C, top two rows compared to bottom two rows). Since backward scanning of 15-17 nt has been observed [69][70][71] , one would expect that buffering would still be predicted in the queuing model even with an increase in 𝑑 𝑠𝑡𝑎𝑙𝑙 of 6 nt. However, our computational modeling predicts that even very fast backward scanning does not restore buffering when 𝑑 𝑠𝑡𝑎𝑙𝑙 is disrupted by 6 nt (Fig.…”