Donna Esposito1,2,3, Julien P.Fey1,4,5,

D.Esposito and J.P.Fey contributed equally to this work Initiation codon context is an important determinant of translation initiation rates in both prokaryotes and eukaryotes. Such sequences include the Shine± Dalgarno ribosome-binding site, as well as other motifs surrounding the initiation codon. One proposed interaction is between the base immediately preceding the initiation codon (±1 position) and the nucleotide 3¢ to the tRNAfMet anticodon, at position 37. Adenine is conserved at position 37, and a uridine at ±1 has been shown in vitro to favor initiation. We have tested this model in vivo, by manipulating the chloroplast of the green alga Chlamydomonas reinhardtii, where the translational machinery is prokaryotic in nature. We show that translational defects imparted by mutations at the petA ±1 position can be suppressed by compen-satory mutations at position 37 of an ectopically expressed tRNAfMet. The mutant tRNAs are fully amino-acylated and do not interfere with the translation of other proteins. Although this extended base pairing is not an absolute requirement for initiation, it may convey added speci®city to transcripts carrying non-standard initiation codons, and/or preserve translational ®delity under certain stress conditions