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Chloroplast Genome

Written By Unknown on Saturday, August 29, 2009 | 12:53 AM

Chloroplast Genome -

The chloroplasts of green plants are cytoplasmic organelles that house the various pigments and enzymes of the light harvesting photosynthetic apparatus. Even before the turn of the century it was clear that green pigmentation was one of the easiest traits to observe in plant breeding experiments. Although some pigmentation traits obeyed Mendel's laws, other colour traits were only transmitted through the female parent that provided the cytoplasm of the zygote.

These observations of cytoplasmic or maternal inheritance eventually led to the hypothesis that chloroplasts must carry genes. We know that chloroplasts contain a unique circular DNA genome that is completely different from the nuclear genome. The presence of a genetic system within chloroplasts had already been inferred from studies on non Mendelian inheritance in 1909, but the presence of organellar DNA and ribosomes was demonstrated only in 1962.

Since then it has been shown that chloroplasts and other plastids contain all the machinery necessary for gene expression. The chloroplast genetic components form a large proportion of those in the leaf, comprising up to 15% of the total DNA and up to 60% of the total ribosomes. The chloroplast genome has been extensively characterized from a variety of species and cooperation between the chloroplast and nuclear genome in chloroplast biogenesis is currently under investigation.

Electron micrographs indicate that the chloroplast DNA is some 10 to 20 times smaller than the E. coli chromosomes. For example, the chloroplast genome of maize (corn) contains 140,000 base pairs of DNA. Such genomes are much too small to encode the approximately 1,000 different proteins found in chloroplasts. Instead, biosynthesis of the chloroplast involves an intimate collaboration between the nuclear and chloroplast genomes.

In fact, every known multimeric protein component of chloroplasts is a mixture of the products of both nuclear and chloroplast genes. Most chloroplast proteins are encoded by nuclear DNA, translated in the cytoplasm, and imported into the chloroplast by a specific transport mechanism that enables polypeptides to cross the outer membrane of the organelle.

However, some 100 chloroplast specific proteins are synthesized within the chloroplast itself. These proteins are encoded by chloroplast DNA, transcribed by the chloroplast specific RNA polymerase, and translated by the chloroplast specific protein-synthesizing machinery. Since RNA cannot cross the outer membrane of the chloroplast, chloroplast ribosomal RNAs and tRNAs must be encoded in chloroplast DNA.

Chloroplasts are not static organelles but can adapt to different physiological conditions, such as high or low levels of light. For example, when grown entirely in the dark, chloroplasts lack chlorophyll but retain carotenoid pigments. Thus many chloroplast genes are light regulated in certain cases by light sensitive promoters.

Structure and Organisation of Chloroplast Genome -
The chloroplast genomes of vascular plants and most algae are quite similar. In general structure and organization, especially by comparison to the wholesale variation seen in the nuclear and mitochondrial genomes. With one Possible exception, all known chloroplast genomes are circular DNA molecules. Size variation is greatest among green algae in which most chloroplast genomes range between about 85 and 300 kb.

The genome of Acetabularia chloroplasts is exceptional in being very large (approximately 2,000 kb) and perhaps composed of linear rather than circular DNA molecules. However, in angiosperms chloroplast genomes in all but two of over 200 species examined are circular and range in size between 120 and 160 kb. The low end of this range is a single group of legumes which lack one copy of the large (15-25 kb) repeated sequence characteristic of most other chloroplast genomes.

Thus the great majority of angiosperm chloroplast genomes actually fall into the relatively narrow range of 135 and 160 kb. Chloroplast DNA (ctDNA) consists of a circular molecule of 83-128 x 106 molecular weight with a size of 1.21-1.93 x 105 bp, which contains about 85% single copy sequences. DNA is present in about 30-200 copies per chloroplast.

A number of genes have been located on the circle and one of the important features is the presence of two copies of the ribosomal DNA sequences. These sequences are often but not always-present on a large inverted repeat. Other genes mapped include those for the large subunit of RuBP. Case, tRNAs, subunits of ATP synthase, and cytochrome.

Most of this size variation can be accounted for by the presence or absence of a portion of the plastid genome which has been duplicated and is present in an inverted orientation in the plastid DNA molecule. The location of this inverted repeat is relatively fixed with respect to other genes and it separates a small single-copy region from a large single copy DNA region.

In most higher plants the inverted repeat is 22 to 26 kbp, within which the rRNA transcription unit is located. In geranium the repeated DNA is larger and genes such as psbB, petB, pelD, petA and rbcL are included in the inverted repeat. Finally, some plastid genomes, such as those in pea and mung bean, lack inverted repeat.

Plastid gene content in higher plants is very constant and many polycistronic transcription units are conserved. Several gene pairs such as psaA­-psaB, psbD-psbC, atpB-atpE, are contranscribed in all the higher plant plas­tid genomes examined to date. The contranscription of genes may ensure that the synthesis of subunits is stoichiometric and or could promote protein-­protein interactions required for assembly of functional complexes.

For example, psaA and psaB encode polypeptides which are tightly associated in the reaction centre of PSI. Other genes, such as rbcL and some gene encoding tRNAs, are not part of polycistronic transcription units. While the plastid gene content of higher plants is very constant, variation in gene order is evident, which results primarily from DNA inversion. DNA.

Inversions have reshuffled plastid genomes such that distances between genes, and relative orientation of transcription units, vary considerably in genomes of higher plants. For example, rps16 is proximal to trnk in barley, whereas in pea rbcL occupies this position. The greatest variation in gene order is found in peas (at least 12 rearrangements), perhaps due to lack of an inverted repeat in this plasmid genome which might stabilize the genome.




DNA Copy Number and Localization -
Multiple copies of the plastid genome are found in each cell and in each plastid as well. The amount of DNA per plastid varies with the stage of leaf and chloroplast development. Proplastids with as few as 22 copies of DNA have been reported, whereas chloroplasts in general contain 200 to 300 DNA molecules. The polyploid nature of the plastid genome is even more striking when one considers leaf cells.
In pea, barley and spinach each natural leaf cell contains 9,000 to 13,000 copies of plastid DNA dispersed in 40 to 120 plastids. The very high effective ploidy of the chloroplast genome means that a significant fraction of the total DNA in a cell as much as 30% if the nuclear genome is small may be of chloroplast origin. This means that rbcL, a single copy plastid gene which encodes the large subunit of Rubisco, is present in about 10,000 copies in a mesophyll cell.
RNA Other Processing Activities -

Although plastid RNAs are neither capped nor polyadenylated, RNA maturation pathways can be very complex. In addition to intron removal, primary transcripts are often cleaved to remove a portion of the untranslated RNA proximal to open reading frames.
The function of the 5' end RNA processing is not known but it may alter stability or transcript translatability. Polycistronic transcripts are also processed at internal sites. Here again the role RNA processing plays gene expression is unclear but one result is differential accumulation of RNA from some parts of long transcription units.

Protein Genes -

Whether or not most chloroplast protein genes are transcribed as parts of operons remains to be determined. It is known that rbcS and psbA produce very abundant monocistronic transcripts but these genes may be exceptions to the general rule. Transcript mapping studies indicate that the situation in other regions of the genome may be much more complex.When Northern blots of electrophoretically separated RNA are probed by hybridization with small cloned fragments of chloroplast DNA, numerous RNA bands with homology to the probe are often seen. Some of the RNA molecules visual-sized in this way are quite large (for example, 4-8 kb), much larger than any one gene, and often many times the length of the probe.

In some cases, it has been shown that most of the RNAs in a series of bands come from the same strand of DNA. Since in angiosperms most chloroplast protein coding genes do not contain introns, this multiplicity of RNAs must reflect the use of multiple initiation or termination sites and or the processing of a long primary transcript.In both cases the initial transcripts are polycistronic and I the production of mature, translatable mRNA must involve processing steps. Many of the intermediate size RNA bands may be processing intermediates of various types.These observations of polycistronic transcripts are surprising since chloroplast protein genes (in contrast to rRNA genes) generally are not organized into the prokaryotic operon pattern in which functionally related genes are closely linked. Some remnants of a prokaryotic operon structure can be discerned in chloroplast genomes, however. One case involves genes for the thylakoid membrane ATPase complex.
Chloroplast Promoter Sequences -

Searches for conserved promoter like sequences upstream from chloroplast genes have revealed elements with considerable homology to bacterial promoter sequences. The E. coli "consensus" promoter sequence contains two conserved regions, one normally found about 35 nucleotides and the other about 10 nucleotides upstream from the start of transcription and referred to as the "35" and "-10" elements respectively;

5'... TTGACA/T... (16-18 nucleotides).. .TATG/AAT... 3'. The chloroplast consensus sequence described by L. Bogorad and his colleagues at Harvard University is as follows: 5' . . . A/g TTG/cA/cNa/t. . . (15-20 nucleotides) . . . T A/tA/tG/aA T. . . 3'. I (In the preceding line, lower case letters represent less frequent alternative bases.)In several cases wherein the start of the RNA transcript has been identified by SI nuclease protection experiments, it occurs within eight nucleotides of the proximal promoter elements. The conservation of these sequence elements and their homology with bacterial promoters support, but does not prove, the notion that they function as chloroplast gene promoters.

Proof requires the direct demonstration that removing or changing these sequences actually affects promoter activity. One way of obtaining such proof is to test altered genes in an in, vitro transcription system.The studies undertaken made progressive deletions of sequences 5' to the gene, moving gradually closer to the start of transcription. Each deletion mutant was characterized by DNA sequencing and then tested for its ability to support accurate transcription. Deletions of sequences further upstream than position -85 had little effect on the production of transcripts, but there was a rapid drop in transcription caused by deletions between -80 and -75.

This region contains the sequence TTGCTTA, the first three nucleotides of which are homologous to the E. coli -35 consensus sequence. There is also a TATAAT sequence between -54 and -59 which is fully homologous to the E. coli -10 consensus sequence. Since transcription was already inhibited by the removal of the sequences further upstream, little effect was seen when the TATAAT sequence was deleted.

Nuclear Genes Encoding Plastid Proteins-

The majority of plastid localized proteins are encoded by nuclear genes. These genes are transcribed by RNA polymerase II and the resultant transcripts are spliced, capped and polyadenylated in the nucleus. The mRNAs then are translated by 80S ribosomes in the cytoplasm to produce proteins which can be transported into plastids posttranslationally.Following uptake into the chloroplast, the proteins are assembled with cofactors and other proteins to form functional complexes, Control sequences at the 5' -end of the gene which are involved in initiating transcription are very similar to the "Pribnow': box and the '-35' region characteristic of bacterial genes.

The mRNA produced from chloroplast genes is not usually polyadenylated, although short sequences up to 20 residues have been reported and, of course, no transport is required as the mRNA is produced in the same compartment as the ribosome on which it will be translated. There is no evidence that chloroplast mRNA is transported but of the chloroplasts and translated on cytoplasmic ribosomes.The tRNA chloroplast population differs distinctly from that in the cytoplasm, as do the aminoacylating enzymes. Ribulose bisphosphate carboxylase is the major protein component of chloroplasts and its synthesis is a good example of cooperation between the genomes. The enzyme consists of eight identical large catalytic subunits encoded by the chloroplast genome and eight identical small regulator subunits encoded in the nucleus.

After synthesis, the large subunit (which has limited solubility) is probably bound by a stabilizing protein to maintain solubility prior to assembly into active enzyme. The small subunit is synthesized in the cytoplasm, on free ribosomes, with an N-terminal leader peptide of about 20 amino acids. The complete peptide is then taken up into the chloroplast in an ATP dependent manner, accompanied by removal of the leader peptide by a stromal peptidase. This mechanism is not analogous to that involving signal peptide cleavage. The synthesis of this important enzyme clearly depends on the coordinate expression of genes in different genomes.
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