A separate reconstruction from this data yielded a map at Most of this additional density stems from chloroplast-unique structures already defined on the unliganded chloroplast ribosome, which are expanded in this antibody-bound map. A few regions of density that do not correspond to densities on the unliganded structure may represent the bound antibody Figure 6 C.
Chloroplast-unique structures near the mRNA entrance channel—via S3, which is involved in helicase activity in bacterial ribosomes [ 45 ], or PSRP-7, which binds mRNA Figure 5 and [ 46 ] —are likely involved in recognizing structured elements in coding regions of chloroplast mRNAs and may act to alter the processivity of translation.
These structures may also be involved in mRNA positioning for translation initiation, in analogy to the mRNA gate structure on the mitochondrial ribosome [ 40 ]. Lowering the threshold visualization levels of either the bound or the unbound maps reveals additional density near the mRNA exit channel, extending up along the head and occluding access to the channel unpublished data , suggesting that alterations in this area must occur to provide mRNAs access to the small subunit of the ribosome.
Chloroplast ribosomes imaged in complex with mRNA, tRNA, and protein factors may be needed to fully resolve structures in this area. Discussion This is the first report of the structure of a chloroplast ribosome, and comes from the organism from which the majority of information on plastid translation has been derived C.
The translation machinery in chloroplasts is clearly based on a prokaryotic-like core, though translation in eukaryotic plastids is more complex than in bacteria from both regulatory and physical perspectives. A large body of research has shown that translation is the key regulated step in chloroplast gene expression [ 49 ].
Structural analysis of the chloroplast ribosome and identification of chloroplast-unique structures on the ribosome provide an important understanding of the physical components utilized for translation regulation in this organelle. Chloroplast-unique structures dominate the solvent-exposed face of the small subunit, and approach both the mRNA entrance and exit channels.
These structures are ideally situated to regulate translation initiation, and genetic and biochemical data suggest that these structures accompany and complement the use of modified S-D sequences and translation initiation mRNP formation. Proteomic studies identified chloroplast-unique ribosomal proteins, primarily on the small subunit of the ribosome [ 26 ] Tables S1 and S2.
The structure presented here allows us to visualize these chloroplast-unique proteins as novel structural domains on the chloroplast ribosome. The primary function of the large subunit of the ribosome is peptide bond formation, and this most basic function of the ribosome has been conserved between eukaryotic, bacterial, and organellar ribosomes.
Here, we confirm this expected structural conservation in the core of chloroplast ribosomes. The small ribosomal subunit is responsible for interactions with mRNAs and initiation factors that position messages for translation initiation [ 21 ], and it also has the duty of quality control in codon decoding during translation. Regions of the small subunit that are responsible for decoding and quality control are structurally conserved with bacterial ribosomes, whereas the chloroplast-unique additions are seen on the small subunit of the ribosome in areas that intersect the path of mRNA during translation initiation, the key regulated step of chloroplast translation.
Taken together, the analyses of two datasets with different sequencing depths suggest that the tissues with greater photosynthetic activity such as the leaf and shoot exhibit higher levels of chloroplast transcripts. While calculating the read distribution for each strand, we found that both the coding and non-coding regions were almost equally covered by all mapped reads Fig.
These findings demonstrate that antisense transcription occurs for both strands of the entire plastome and is most likely associated with long non-coding RNAs lncRNAs transcription Figs 2A and 3 Figure 2: Both strands of the Arabidopsis plastome were transcribed. A Strand-specific transcriptome reads were mapped to both strands of the Arabidopsis plastome.
The outer and third tracks represent genes in the outer and inner strand, respectively. The black histograms of the second and fourth tracks indicate RNAseq reads mapping scale logtransformed numbers of sequence reads per nucleotide. B Comparisons of intergenic and coding region transcription for each strand of the Arabidopsis plastome.
Full size image Figure 3: Antisense transcription in the chloroplast genome. Strand-specific transcriptome reads showing that antisense transcription light blue exceeds sense transcription light red for the ndhC gene. Full size image Exclusion of nuclear-localized plastid DNAs nupDNAs transcription NupDNA fragments were thought to be quite common in plant nuclear genomes, and they should be non-functional, indicating that they would be rapidly fragmented and eliminated from the nuclear genome during evolution 27 , The transcriptome data in the present study were generated from whole-cell preparations, providing the possibility that some transcriptome reads may come from the nupDNA transcripts.
We counted the reads depth at the positions that were variable between nupDNAs and the chloroplast reference genome. The reads depths of the regions that contain variable positions or junctions were significantly lower and close to zero compared to those covering non-variable positions and the corresponding chloroplast genomic regions Fig.
Besides, a plant cell often harbors hundreds of chloroplast genomes to 1, chloroplast genome in a leaf cell 29 , therefore, when sequence reads of hundreds of high quality chloroplasts are aligned, the nupDNA transcripts, if present, can be neglected.
Figure 4: Examination of nupDNA transcription. All nupDNAs and plastome homologous sequences had a sequence length of bp x-axis. The y-axis represents the RNAseq reads mapping depth per nucleotide.
Full size image Moreover, the above-mentioned rice tissue-specific reads mapping results showed that after sequence reads normalization and rRNA depletion, the mapped plastid transcriptome reads were 0. Among the studied species, the rice genome exhibited the largest proportion of nupDNAs 27 , However, both nupDNA and tissue-specific transcription patterns indicate that the p-transcriptome reads mapping results reflect the actual plastome transcription.
The entire genome transcription of cyanobacteria Cyanobacteria are prokaryotes thought to be related to the evolutionary ancestors of the chloroplasts 8 , To investigate whether full transcription of the algae and plant plastomes was derived from cyanobacteria 30 , we analyzed three cyanobacteria with high-quality reference genomes and high-throughput transcriptome datasets: Synechocystis sp.
PCC , Synechococcus sp. PCC , and Prochlorococcus marinus subsp. Even though their genome sizes varied from 1. These reads were nearly evenly mapped to both coding and non-coding regions Fig. Thus, cyanobacteria genomes may share the same transcription mechanism with plant plastomes, indicating a common ancestral origin of transcription.
Figure 5: Full transcription of the cyanobacteria genomes. A—C Maps of the cyanobacteria genomes transcription with the outer and third tracks representing genes in the genome, and the black histogram of the second track represent RNAseq reads mapping scale logtransformed numbers of sequence reads per nucleotide. D Comparisons of intergenic and coding region transcription for the five species.
Box-and-whisker plots in which the whiskers denote the 5th and 95th quantiles of log2-transformed numbers of sequence reads per nucleotide are shown for all the intergenic sequences NonCDS and coding sequences CDS. Chloroplast RNA editing was hypothesized to have evolved simultaneously with the origin of the first land plants 31 because it was poorly observed in plastid-encoded RNAs of algae groups 8.
By further examining the reads mapping results of the transcriptomes, we detected 91, , and 51 RNA editing sites in the rice, maize, and Arabidopsis plastomes, respectively Supplementary Table S6. Moreover, 69 and 75 editing sites were found in Chlamydomonas and C.
Interestingly, only 6, 15, and 43 editing sites were observed in P. Some genes involved in photosynthetic metabolism e. While, conserved editing sites within these genes from these examined species were quite spare, this may be partially owing to frequent gene sequence variation among them. Thus, our results support the hypothesis that RNA editing emergence preceded chloroplast endosymbiosis De novo plastome assembly from transcriptome data The evidence for whole-genome transcription suggests that the entire genome can be transcribed into RNAs.
Conversely, this finding implies that the plastome sequence can be straightforwardly assembled from the transcriptome. The complete plastomes were de novo assembled from these species, which included 2 bryophytes and 12 angiosperms Fig. This added an extra layer of evidence for whole-plastome transcription in photosynthetic eukaryote chloroplasts. Figure 6: Complete cp genomes were de novo assembled from transcriptome data.
The wrap sequence alignment of the assembled genome. The black blocks depict genome similarity for these species. A detailed species list is provided in Supplementary Table S7. Full size image A multiple arrangement transcription model It has long been thought that some plastome genes were transcribed via typical polycistronic operon transcriptional model as observed in Escherichia coli 8 , Recently, a novel genome-wide transcriptional start site TSS category assignment was reported in both chloroplast and cyanobacterial genomes 14 , 33 , 34 , which identified numerous promoters inside open reading frames ORFs , non-coding regions, antisense to known genes, and genomic regions without any predicted genes.
These functional TSSs far exceeded the numbers of genes within gene clusters 14 , 33 , Considering the extensive transcription initiation and infrequent and stochastic termination described above and the observed full transcription of the plastomes Fig. This generates numerous overlapping precursor transcripts with variable sizes that cover both strands of the entire genome.
Because the precursor transcripts are likely to be transcribed from various combinations of start and termination sites, many transcripts can include incomplete ORFs and pseudogenes Figure 7: Model for the full plastome transcription and procession. A Transcription initiation of a gene cluster occurs from multiple promoters bent arrow upstream of open reading frames ORFs or within ORFs. Together with inefficient transcription termination, this setup generates numerous precursor transcripts that can include complete or incomplete ORFs.
Introns and RNA stem—loop structures are depicted as light black rectangles and hairpins, respectively. B Precursor transcripts are processed by a combination of exo- and endo-ribonucleases. Introns and incomplete ORFs without sequence-specific RNA-binding proteins protection were digested by exo- or endo-ribonucleases. Small RNA transcriptome reads of four tissues were mapped to the rice plastome.
The colored histograms represent small RNA mapping coverage in a logarithmic scale. Detailed statistics of reads mapping is given in Supplementary Table S8. Full size image The model presented here can feasibly explain the large RNA transcription outputs in algae and plant plastomes, possibly also in cyanobacteria.
Previous studies have genome-wide identified numerous transcriptional start sites TSS in both chloroplast e. Thus, the mechanism of plastome transcription proposed in such a model may not be confined by intrinsic gene transcriptional initiation and termination. Multiple transcription initiation and termination form the basis for full transcription of the plastomes.
B Comparisons of intergenic and coding region transcription for each strand of the Arabidopsis plastome. Relatively few of the data sets involve organisms whose growth rate is determined by P supply. Our findings indicate a complex prokaryotic genome regulation when processing primary transcripts. Box-and-whisker plots in which the whiskers denote the 5th and 95th quantiles of log2-transformed numbers of sequence reads per nucleotide for all intergenic sequences NonCDS and coding sequences CDS. Figure 7: Model for the full plastome transcription and procession. Eukaryotic and bacterial growth regulation systems of independent origins work in unison to control chloroplast rRNA transcription.
This means that there is g per mol ribosomal RNA in eukaryotes Loladze and Elser, , or 1. Each of these systems has highly regulated translation initiation, which suggests that structural adaptation to the ribosome for specialized translation regulation may be quite ubiquitous in nature. Full plastome transcription may constitute a new level of prokaryotic genome transcriptional regulation at the level of processing of primary transcripts. This can be related to the finding by Zhu et al.
The small subunit of the ribosome has evolved and adapted as a means to regulate translation initiation, whereas the large subunit of the ribosome is more evolutionarily stable, maintaining the basic function and integrity of the core reactions of peptide bond formation and nascent peptide delivery. For all the examined species, intergenic regions were also hit by substantial sequence reads, only slightly lower than that for coding regions Fig. Thus, the results of this study lay the foundation for future research in chloroplast ribosome biogenesis. Moreover, 69 and 75 editing sites were found in Chlamydomonas and C. The transcriptional diversity of RNAs together with further posttranscriptional processes generates uncountable plastome transcripts
These values are significantly higher than the highest values cited by Karpinets et al. D Comparisons of intergenic and coding region transcription for the five species. The other conclusion is that total rRNA decreased after full expansion, despite the absence of net protein synthesis, presumably because RNA is needed for protein turnover. Other aspects of genetically determined variations in growth rate among algae are considered by Flynn The primary function of the large subunit of the ribosome is peptide bond formation, and this most basic function of the ribosome has been conserved between eukaryotic, bacterial, and organellar ribosomes. Such a model for bacterial translation is quite simple as in Figure 1 : mRNAs, even as they are being transcribed, are positioned via S1 binding and the S-D interaction with their start site AUG in the P-site of the ribosome ready for initiation.
An obvious possibility is greater fractional extractability of protein than of RNA from the photolithotrophs than the chemoorganotrophs.