The effects of codon context on in vivo translation speed

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.