Short translational ramp determines efficiency of protein synthesis

Abstract

It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence 1-4 . These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5' translational ramp, likely contributes to the efficiency of protein synthesis 5-9 . Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias 6,9 , the rate of translation initiation 10-15 , mRNA and ribosome structure 11,12,14,[16][17][18] , or retention of initiation factors during early elongation events 19 .Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4 th or 5 th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

Inferring efficiency of translation initiation and elongation from ribosome profiling

“…This result is consistent with two recent studies that stressed the importance of the first 10 codons in regulating protein production (Chu et al, 2014;Szavits-Nossan et al, 2018a). Furthermore, the first codons are also experimentally recognised to be critical in determining protein synthesis (Kudla et al, 2009;Bentele et al, 2014;Cambray et al, 2018;Verma et al, 2019). Finally, this result is also consistent with the "slow ramp" hypothesis (Tuller et al, 2010): a ribosome translating the first (slower) codons will be less prone to interfere with other ribosomes downstream the sequence.…”

Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

“…The strong biases on disfavored DE/P-rich sequences and favored bulky amino acids in the N-terminal regions of bacterial ORFs are in agreement with this assumption (Figure 4 and Figure S4). Many previous studies have pointed out that the sequence features just adjacent to the start codon significantly influence the efficiency of protein biosynthesis (Allert et al, 2010; Goodman et al, 2013; Kudla et al, 2009; Osterman et al, 2020; Salis et al, 2009; Sprengart et al, 1996; Verma et al, 2019). Considering the “bulkiness” of the nascent polypeptide when interpreting the datasets might disclose new landscapes from previous studies.…”

Biofoundry-assisted expression and characterisation of plant proteins

“…Our workflows were aided by the use of the high-affinity, 11 amino acid HiBiT tag (87), which is able to complex with a 18 kDa engineered NanoLuc polypeptide (88) and luminesce proportional to the level of HiBiT-tagged protein. We found that this worked well as a C-terminal tag but reduced yields when fused to the N-terminus of sfGFP ( Figure 2 ), perhaps because the HiBiT amino acid sequence is not optimal for early translational elongation (89). Although sfGFP has been optimised to improve folding (90), the minimal size of the HiBiT tag may be less likely to inhibit protein function and accessibility.…”