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20.11 Chloroplast DNA of rice.

photosynthesis, and several proteins having roles in nonpho-tosynthesis processes. A key protein encoded by cpDNA is ribulose-1,5-bisphosphate carboxylase-oxygenase (abbreviated RuBisCO), which participates in carbon fixation of photosynthesis. RuBisCO makes up about 50% of the protein found in green plants and is therefore considered the most abundant protein on earth. It is a complex protein consisting of eight identical large subunits and eight identical small subunits. The large subunit is encoded by chloroplast DNA, whereas the small subunit is encoded by nuclear DNA.

The circular chloroplast genome has genes on both of its strands. Some chloroplast genes have been identified on the basis of a start and stop codon in the same reading frame, but no protein products have yet been isolated for these genes. These sequences are referred to as open reading frames. A prominent feature of most chloroplast genomes is the presence of a large inverted repeat. In rice, this repeat includes genes for 23S rRNA, 4.5S rRNA, and 5S rRNA, as well as several genes for tRNAs and proteins (see Figure 20.11). In some plants, these repeats include the majority of the genome, whereas, in others, the repeats are absent entirely. Much of cpDNA consists of noncoding sequences, and introns are found in many chloroplast genes. Finally, many of the sequences in cpDNA are quite similar to those found in equivalent eubacterial genes.

Concepts]"

Most chloroplast genomes consist of a single, circular DNA molecule not complexed with histone proteins. Although there is considerable size variation, the cpDNAs found in most vascular plants are about 150,000 bp. Genes are scattered in the circular chloroplast genome, and many contain introns. Most cpDNAs contain a large inverted repeat.

Replication, Transcription, and Translation of cpDNA

Little is known about the process of replication of cpDNA. The results of studies viewing cpDNA replication with electron microscopy suggest that replication begins within two D loops and spreads outward to form a theta-like structure. After an initial round of replication, DNA synthesis may switch to a rolling-circle-type mechanism (see Figure 12.5).

The transcription and translation of chloroplast genes are similar in many respects to these processes in eubacteria. For example, promoters found in cpDNA are virtually identical with those found in eubacteria and possess sequences similar to the —10 and —35 consensus sequences of eubacterial promoters. The same antibiotics that inhibit protein synthesis in eubacteria (as well as in mitochondria) inhibit protein synthesis in chloroplasts, indicating that protein synthesis in eubac-teria and chloroplasts is similar. Chloroplast translation is initiated by N-formylmethionine, just as it is in eubacteria.

Most genes in cpDNA are transcribed in groups; only a few genes have their own promoters and are transcribed as separate mRNA molecules. The RNA polymerase that transcribes cpDNA is more similar to eubacterial RNA poly-merase than to any of the RNA polymerases that transcribe eukaryotic nuclear genes. Like eubacterial mRNAs, chloro-plast mRNAs are not capped at the 5' ends, and poly(A) tails are not added to the 3' ends. However, introns are removed from some RNA molecules after transcription, and the 5' and 3' ends may undergo some additional processing before the molecules are translated. Like eubacterial mRNAs, many chloroplast mRNAs have a Shine-Dalgarno sequence in the 5' untranslated region, which may serve as a ribosome-binding site.

Chloroplasts, like eubacteria, contain 70S ribosomes that consist of two subunits, a large 50S subunit and a smaller 30S subunit. The small subunit includes a single RNA molecule that is 16S in size, similar to that found in the small subunit of eubacterial ribosomes. The larger 50S subunit includes three rRNA molecules: a 23S rRNA, a 5S rRNA, and a 4.5 rRNA. In eubacterial ribosomes, the large subunit possesses only two rRNA molecules, which are 23S and 5S in size. The 4.5S rRNA molecule found in the large subunit of chloroplast ribosomes is homologous to the 3' end of the 23S rRNA found in eubacteria; so the structure of the chloroplast ribosome is very similar to that of ribo-somes found in eubacteria.

Initiation factors, elongation factors, and termination factors function in chloroplast translation and eubacterial translation in similar ways. Most chloroplast chromosomes encode from 30 to 35 different tRNAs, suggesting that the expanded wobble seen in mitochondria does not exist in chloroplast translation. Only universal codons have been found in cpDNA, and translation in chloroplast starts with N-formylmethionine as the first amino acid.

Evolution of cpDNA

The DNA sequences of chloroplasts are very similar to those found in cyanobacteria; so chloroplast genomes clearly have a eubacterial ancestry. Overall, cpDNA sequences evolve slowly compared with sequences in nuclear DNA and some mtDNA. For most chloroplast genomes, size and gene organization are similar, although there are some notable exceptions.

Concepts]"

Many aspects of the transcription and translation of cpDNA are similar to those of eubacteria. Chloroplast DNA sequences are most similar to DNA sequences in cyanobacteria which supports the endosymbiotic theory. Most cpDNA evolves slowly in sequence and structure.

www.whfreeman.com/pierce Information on chloroplast genomes that have been sequenced

Connecting Concepts

Genome Comparisons

A theme running through the preceding discussions of mitochondrial and chloroplast genomes has been a comparison of these genomes with those found in eubacterial and eukaryotic cells (Table 20.4). The endosymbiotic theory indicates that mitochondria and chloroplasts evolved from eubacterial ancestors, and one might therefore assume that mtDNA and cpDNA would be similar to DNA found in eubacterial cells. The actual situation is more complex: mitochondrial DNA and chloroplast DNA possess a mix of eubacterial, eukaryotic, and unique characteristics.

The mitochondrial and chloroplast genomes are similar to those of eubacterial cells in that they are relatively small, lack histone proteins, and are usually on circular DNA molecules. Gene organization and the expression of organelle genomes, however, display some similarities to eubacterial genomes and some similarities to eukaryotic genomes. Introns are present in some organelle genomes but are absent from others. Pre-mRNA introns (see p. 000 in Chapter 14 for a discussion of different types of introns) are absent from mitochondrial and chloroplast genes, as they are from eubacterial genes. Group II introns are present in some organelle and eubacterial genomes but are absent from eukaryotic nuclear genomes. Group I introns are common in some mtDNA and in most cpDNA, and these introns are also found in eubacterial, archaeal, and eukaryotic genomes.

Polycistronic mRNA, which is common in eubacteria but uncommon in eukaryotes, is also found in mitochondria and especially chloroplasts. Human mtDNA, which has little noncoding DNA between genes and little repetitive DNA, is similar in organization to that of typical eubacterial chromosomes, but other mitochondrial and chloroplast genomes possess long noncoding sequences between genes.

Antibiotics that inhibit eubacterial translation also inhibit organelle translation, and the 5' cap, which is added to eukaryotic mRNA after transcription, is absent from organelle mRNA. A 3' poly(A) tail, characteristic of most nuclear mRNAs, is present only in some animal mitochondrial mRNA, and it appears to be fundamentally different from that found in nuclear mRNAs. Shine-Dal-garno sequences, the ribosome-binding sites characteristic of eubacterial DNA, are present in some cpDNA but are absent in mtDNA. Finally, some mitochondrial genomes use nonuniversal codons and have extended wobble, which is rare in both eubacterial and eukaryotic DNA.

What conclusions can we draw from these comparisons? Clearly, the genomes of mitochondria and chloroplasts are not typical of the nuclear genomes of the eukaryotic cells in which they reside. In sequence, organelle DNA is most similar to eubacterial DNA, but many aspects of organization and expression in organelle genomes are unique. It is important to remember that the endosymbi-otic theory does not propose that mitochondria and chloroplasts are eubacterial in nature but that they arose

Comparison of nuclear eukaryotic, eubacterial, mitochondrial, and chloroplast genomes

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