In vivo evidence for the prokaryotic model of extended codon-anticodon interaction in translation initiation

Abstract

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 tRNAf Met 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 compensatory mutations at position 37 of an ectopically expressed tRNA fMet . The mutant tRNAs are fully aminoacylated 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.

Regulatory Sequences of Orthologous petD Chloroplast mRNAs are Highly Specific among Chlamydomonas Species

“…In petA, a U to A substitution one nt upstream of the initiation codon had no detectable affect on translation with the canonical AUG (Chen et al, 1995). When combined with the non-canonical AUU initiation codon the same U to A substitution still supported translation, but at a rate that was reduced by about fivefold (Esposito et al, 2003). Based on this, the U to A substitution in C. eugametos one nt upstream of the canonical AUG initiation codon by itself is likely to have little effect on translation in C. reinhardtii.…”

“…Substitutions in C. reinhardtii petD and petA translation initiation context sequences can affect translation when combined with non-canonical initiation codons (Chen et al, 1995;Esposito et al, 2003). In petA, a U to A substitution one nt upstream of the initiation codon had no detectable affect on translation with the canonical AUG (Chen et al, 1995).…”

Development of Novel Types of Plastid Transformation Vectors and Evaluation of Factors Controlling Expression

“…Most plastid proteins are processed at the N-terminus (Giglione & Meinnel, 2001) whereby the life-span of the mature proteins is influenced by the N-terminal amino acid composition (Giglione et al, 2003). In addition, extended base pairing with the fMet-tRNA anticodon affects translation in chloroplasts (Esposito et al, 2003). A plastid mRNA containing U at pos.…”

Chloroplast translation regulation

“…Additionally, the initiation codon context has been shown to affect translation in a limited set of mRNAs. In the case of petA mRNA, the underlying mechanism is mediated through an extended codon-anticodon interaction between a relatively conserved adenine at the -1 position in the mRNA and the nucleotide at position 37 on the 3¢ side of the tRNA (Met) anticodon (Esposito et al 2003). Table 1 summarizes the chloroplast mRNAs for which interacting nuclear-encoded factors affecting translation are known, and shows schematically those cis-acting elements within the 5¢ UTR that have been described to be important for translation or mRNA stability.…”