Abstract
Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.
Highlights
Translation initiation is a major rate-limiting step for protein synthesis
To decipher how mRNA sequence and its encoded peptide influence protein synthesis efficiency, we focused on the region surrounding the +10 nucleotide position in an engineered GFPreporter sequence
In summary, we show that the efficiency of protein synthesis is strongly dependent on the nucleotide sequence positions 7–15 and the resulting amino acid positions 3–5 in the nascent peptide, in addition to the overall mRNA structure and codon content
Summary
Translation initiation is a major rate-limiting step for protein synthesis. recent studies strongly suggest that the efficiency of protein synthesis is regulated by multiple factors that impact the elongation phase. We report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Potential factors that contribute to protein synthesis efficiency have been discovered using both endogenous genes and reporter sequences by focusing on tRNA abundance, amino acid sequence or both mRNA sequence and structure[6,7,8,9,10,11,12,13,14,15,16,17,18,19]. Reduced abundance of tRNAs coding for N-terminal protein residues may play a crucial role in slowing down initial rounds of translation elongation[9,14,21] Such a translational ramp would be beneficial in preventing detrimental collision-dependent abortion of protein synthesis[14,18,22]. We present data that strongly suggest that the mRNA and the encoded protein sequences of the first five codons are key in dictating the efficiency of protein synthesis
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