Synonymous Codons: Choose Wisely for Expression

Abstract

The genetic code, which defines the amino acid sequence of a protein, also contains information that influences the rate and efficiency of translation. Neither the mechanisms nor functions of codon-mediated regulation were well understood. The prevailing model was that the slow translation of codons decoded by rare tRNAs reduces efficiency. Recent genome-wide analyses have clarified several issues. Specific codons and codon combinations modulate ribosome speed and facilitate protein folding. However, tRNA availability is not the sole determinant of rate; rather, interactions between adjacent codons and wobble base pairing are key. One mechanism linking translation efficiency and codon use is that slower decoding is coupled to reduced mRNA stability. Changes in tRNA supply mediate biological regulationfor instance,, changes in tRNA amounts facilitate cancer metastasis.

Keywords: codon pair; mRNA decay; protein folding; ribosome profiling; synonymous codons; translation.

Figure I. Schematic of the steps in ribosome profiling

To obtain ribosome footprints, cell lysates, obtained from cells in which translation has been rapidly stopped, are treated with nucleases to digest unprotected RNAs. Ribosome-protected fragments of discrete sizes (22–28 nucleotides in eukaryotes and 15–45 nucleotides in bacteria) are isolated, converted to a library and subjected to deep sequencing. To obtain information on the identity and amounts of mRNA, mRNA is subjected to random fragmentation, isolated, converted to a similar library and sequenced. Sequences are aligned to the genome, such that codons in the A, P and E sites of the ribosome are distinguished. As expected, most ribosome footprints (blue) map to sequences that encode proteins (beginning with the AUG initiation codon and ending at the stop codon) and exhibit a 3 nucleotide periodicity (shown to right of ribosome reads); mRNA footprints (red) extend outside of the translated regions and do not exhibit this periodicity. UTR: untranslated region; ORF: open reading frame.

Figure 1. Schematic of translation elongation

The top diagram shows an elongating ribosome, including the 3 sites for tRNA binding (A, P, and E) that span the large and small ribosomal subunits; the nascent polypeptide (which is attached to the P site tRNA and exits through the exit tunnel in the large subunit); and the mRNA (which enters and exits through the small subunit, moving 5’ to 3’). Bacteria specific components are shown in parentheses. First, addition of a new amino acid begins with the delivery, recognition and accommodation of an aminoacyl-tRNA into the A site of the ribosome. In this step, GTP-bound elongation factor (eEF1A GTP in eukaryotes or EF-Tu GTP in bacteria) delivers an aminoacyl-tRNA to the A site of the ribosome, where base pairing interactions between the anticodon of the tRNA and the 3 bases of the codon in the mRNA trigger hydrolysis of GTP, release of the deacylated tRNA from the E site of the ribosome, release of the GDP-bound elongation factor and further accommodation of the tRNA (a second proofreading step that depends upon codon-anticodon base pairing). Next, the addition of the amino acid to the growing polypeptide requires peptide bond formation, which involves close approximation of the 3’ ends of the aminoacyl and the peptidyl site tRNAs in the peptidyl transferase center of the large subunit, and large scale movement of the ribosomal subunits relative to each other to form a hybrid state. Finally, to complete the process, translocation of the mRNA (with its associated tRNAs) involves another GTP-bound elongation factor (eEF2 GTP in eukaryotes or EF-G GTP in bacteria), and large scale ribosome movement to restore the classical state. This process is repeated until the ribosome encounters a termination codon.

Figure 2. The consequences of codon choice

Effects of different types of codon choice. (A) Genes encoded primarily with optimal codons (blue) are rapidly and efficiently translated. (B) Genes encoded primarily with suboptimal or slowly decoded codons (orange) are inefficiently translated; their mRNAs exhibit decreased stability, mediated in yeast by Dhh1 protein. (C) Inhibitory codon pairs (red box) are very slowly decoded, reduce protein output significantly, and sometimes recruit quality control machinery. (D) The efficiency of translation initiation influences the magnitude of the effects of suboptimal codon use on expression (ratio of expression of synonymous variants encoding the same protein). (E) A change in the amount of two specific tRNAs drives cancer metastasis and modulates expression of specific genes. (F) Harmonized codon use, a combination of rapidly and slowly decoded codons, can facilitate co-translational protein folding to obtain biologically active molecules.