Identification of functional lox sites in the plastid genome

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.     

Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.     

Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs

Many of the chloroplast mRNAs possess Shine-Dalgarno (SD)-like sequences (typically GGAGG) in the 5'-untranslated regions, but the position is highly variable. Using a homologous in vitro translation system, we assessed the role for translation of SD-like sequences in four tobacco chloroplast mRNAs. The rbcL mRNA has a typical SD-like sequence at a position similar to the conserved position (-12 to -4 with respect to the start codon) observed in E. coli, and this sequence was found to be essential for translation. This was also the case for the atpE mRNA. However, SD-like sequences in the rps12 mRNA and in the petB mRNA is located far from (-44 to -42) and too close to (-5 to -2) the initiation codon, respectively, and these sequences were not essential for translation. These results indicate that functional SD-like sequences are located around 10 nucleotides upstream from the translational start codon. Competition assays confirmed that a functional SD-like sequence interacts with the 3' terminus of chloroplast 16S rRNA.     

Cost minimization of ribosomal frameshifts

Properties of mRNA leading regions that modulate protein synthesis are little known (besides effects of their secondary structure). Here I explore how coding properties of leading regions may account for their disparate efficiencies. Trinucleotides that form off frame stop codons decrease costs of ribosomal slippages during protein synthesis: protein activity (as a proxy of gene expression, and as measured in experiments using artificial variants of 5' leading sequences of beta galactosidase in Escherichia coli) increases proportionally to the number of stop motifs in any frame in the 5' leading region. This suggests that stop codons in the 5' leading region, upstream of the recognized coding sequence, terminate eventual translations that sometimes start before ribosomes reach the mRNA's recognized start codon, increasing efficiency. This hypothesis is confirmed by further analyses: mRNAs with 5' leading regions containing in the same frame a start preceding a stop codon (in any frame) produce less enzymatic activity than those with the stop preceding the start. Hence coding properties, in addition to other properties, such as the secondary structure of the 5' leading region, regulate translation. This experimentally (a) confirms that within coding regions, off frame stops increase protein synthesis efficiency by early stopping frameshifted translation; (b) suggests that this occurs for all frames also in 5' leading regions and that (c) several alternative start codons that function at different probabilities should routinely be considered for all genes in the region of the recognized initiation codon. An unknown number of short peptides might be translated from coding and non-coding regions of RNAs.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.