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

Abstract

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 specificity to transcripts carrying non-standard initiation codons, and/or preserve translational fidelity under certain stress conditions.

Fig. 1. Expression of ectopic tRNAs in the chloroplast. (A) Extended interaction model between the initiator tRNA and mRNA _–1U. The three closed circles denote the tRNA anticodon; the A₃₇ residue is shown by a hatched circle. (B) Transformation strategy to mutate the petA gene and, for certain constructs, to introduce an additional tRNA gene into the chloroplast genome. The top part shows the location of the endogenous trnfM gene downstream of psbC, and its distance from the petA–petD region. The lower line shows the altered region in transformants (the 1.8 kb aadA/petD intergenic region is not to scale), with gene orientations indicated by arrows. The notation trnfM* indicates the ectopic copy. (C) Names of the strains created (left column), and the number of predicted base pairs that the particular version of the petA mRNA would form with the endogenous tRNA^fMet (middle column) or with the ectopic tRNA^fMet where present (right column). Strains are named by the petA mRNA sequences at –1 and the initiation codon (e.g. U-AUG), plus position 37 of the tRNA^fMet (e.g. +A). Mutated residues relative to WT are in bold. In columns 2 and 3, the strongest base pairing possibility is bold and underlined.

Fig. 2. Expression and aminoacylation of tRNAs. RNAs from the strains indicated above the gels were extracted at pH 5.5 or 9.5, as noted below the gels, and fractionated by electrophoresis through a 15% polyacrylamide gel buffered to pH 5.5. Gels were transferred to membranes and probed with oligonucleotides complementary to (A) tRNA^Leu and WT tRNA^fMet, as indicated at the right, or (B) tRNA^Leu and the ectopic U₃₇ tRNA^fMet. The positions of charged (+Leu and +fMet) and uncharged (–Leu and –fMet) tRNAs are indicated at the left.

Fig. 3. Chloroplast protein pulse labeling in the presence of a cytoplasmic translation inhibitor. Strains are indicated across the top with nomenclature as described in the legend to Figure 1; ΔpetA is a strain lacking the petA gene. Clearly visible chloroplast proteins are indicated at the left: PsaB, core subunit B of photosystem I (PSI); LS, large subunit of Rubisco; ATPase β, β subunit of the ATP synthase; ATPase α, α subunit of the ATP synthase; apo-CP47: 47 kDa subunit of PSII; apo-CP43, 43 kDa PSII subunit; cytf, cytochrome f; D2, PSII reaction center; D1, PSII reaction center; SUIV, subunit IV of the cytochrome b₆/f complex. The lower panel shows a section of the same gel, with the exposure adjusted to emphasize cytf synthesis.

Fig. 4. Analysis of cyt f accumulation. Immunoblot analysis was used to measure the accumulation of cyt f (34 kDa) in the indicated strains, with the ATPase β subunit (55 kDa) as a loading control. The values indicated for cyt f accumulation, shown beneath each gel and relative to WT, were estimated from multiple blots. (A) Control strains compared with those with mRNA –1 mutations. (B) Control strains compared with those with a WT –1 sequence in petA, but expressing ectopic tRNAs. (C) A WT control compared with strains carrying _–1C mutations in petA mRNA. Here the indicated cyt f accumulation levels are mean values obtained for four independent clones for each strain, quantified against both the ATPase β subunit and the OEE2 protein. OEE2 is the nucleus-encoded PSII oxygen-evolving enhancer protein 2 (Mayfield et al., 1987).