Abstract
We developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Low frequency effects of so-called “silent” codon changes and codon neighbor (context) effects could be measured using this assay. An advantage of this system is that translation speed is unaffected by the primary sequence of the His leader peptide. We show that the apparent speed at which ribosomes translate synonymous codons can vary substantially even for synonymous codons read by the same tRNA species. Assaying translation through codon pairs for the 5′- and 3′- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including Shine-Dalgarno sequence, rate of dipeptide bond formation, codon context, and charged tRNA levels.
Highlights
Synonymous mutations are DNA sequence changes that do not affect the protein amino acid sequence
The central dogma of molecular biology states that DNA is transcribed into RNA, which is translated into protein
The process of translation from messenger RNA into protein by the ribosome is a complicated process involving transfer RNA intermediates with attached amino acids that must recognize 3 base codons in the mRNA sequence to be translated with the correct amino acid
Summary
Synonymous mutations are DNA (or mRNA) sequence changes that do not affect the protein amino acid sequence. The expression of foreign proteins in E. coli is dramatically affected by the presence or absence of rare codons in the coding sequence. The ability of a nonsense suppressor tRNA to recognize a stop codon was dramatically affected by the adjacent codon [7]. This effect was in the context of recognition of three bases by the tRNA and not four [8]. Similar results were later observed with suppression of missense mutations [9,10,11] These studies provided in vivo genetic evidence for context effects on translation
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