Gene Expression of ChloroplastChloroplast DNAs are usually a circular double stranded DNA molecules containing a long single copy section (LSC) and a short single copy section (SSC) separated by two inverted repeats. Normally chloroplast genome (cp genome) contains about 100- 200 base pairs and codes for about 60 proteins 30tRNAs and 4rRNA in a higher plant. Some of these genes encode components such as RNA polymerase, ribosomal proteins, tRNAs, and rRNAs which are involved in chloroplast gene expression machinery and subunits of photosynthetic enzymes such as Rubisco, PSII, the cytochrome b6f complex, PSI, ATP synthase and NADH dehydrogenase. Some genes encode proteins like ClpP, AccD, Ycf1, and Ycf2 which are involved in other metabolic processes. Most of the proteins with in chloroplast are nuclear gene products that are synthesized in the cytosol and , imported and some are synthesized within chloroplast. RuBisCO (Ribulose-1, 5-bisphosphate carboxylase/oxygenase) the major enzyme involved in carbon fixation process and photo respiration within chloroplast. Nuclear gene (rbcS) encodes the small subunit, whereas a single gene (rbcL) located on multiple copies of DNA in the chloroplast encodes the large subunit. Aassembly is occurred correctly in the presence special folding proteins called chaperonins.Most of the cp genes are arranged in operon-like units and co-transcribed. For examples psbD-psbC gene cluster is an operon and in psbD-psbC gene transcription, a different (closer) promoter is used in the presence of the light called light-responsive promoter (LRP). Nearly there are 30 promoters in higher plant cp DNA and a "transcription unit" is determined by the position of the promoter (5') and terminator (3') signals. Most of the promoters resembles the major E. coli σ70 (or -10,-35) promoter. In some promoters the distance between -10 and -35 regions is critical. For sofme genes, no -10 and -35 regions as they have other promoter type. The distance between -10 and start point (+1) is less critical. The consensus sequences at -10,-35 and +1 are highly variable among species. Terminators not clearly defined, but tRNA genes seem to be transcription terminators. Transcription regulation is done mainly by the regulation of posttranscriptional steps and also at initiation step using alternative promoters. Transcription rate is affected due to the presence of some genes (psbD) by upstream sequences that bind regulatory proteins and the “promoter strength”.There are 2 major CP RNA polymerases in vascular plants. They are E. coli or eubacterial-like polymerase (PEP, plastid-encoded polymerase) and phage-like polymerase (NEP, nuclear-encoded polymerase). PEP is composed of core and Sigma factor. Core is made up of 4 subunits, α2 ββ' (α is encoded by the rpoA gene, β is encoded by the rpoB gene, β' is encoded by the rpoC1 and rpoC2 genes). Sigma factor is needed to initiate transcription as it recognizes -10,-35 regions for some promoters. This polymerase is nhibited by rifampicin. NEP polymerase consisted of Catalytic subunit is similar to the 1-subunit phage (e.g., T7) and mitochondrial RNA polymerases and it is coded by a nuclear gene. It identifies the promoter consisted of a single region of 7-10 bp. It is insensitive to rifampicin. NEP is more important early in plastid development, when plastid transcription and translation is relatively low. This transcribes rRNA, rpo and other genetic functions genes (GFG).PEP is more important in mature chloroplasts as it transcribes some GFG genes and most of photosynthesis genes.CP pre-mRNA is processed by cleavage(s) or splicing or both. CP mRNAs are not polyadenylated, and are not capped. Nucleolytic Cleavages are occurred in 2 ways. Endonucleases cut internal regions specifically (e.g. in between genes) and exonucleases trim at 3' or 5'-ends, processively less specifically. Inverted repeats (IR) occur at 3'-end of most cp protein encoding genes also they act as processing sites, determining the 3'-end of mRNAs. Proteins recognize the 3'-IR, bind and stop the processive exonucleases. An

endonuclease cleaves at the 3’-IR. Translation machinery is similar to bacteria. Ribosomes are 70S (composed of L (50S) and S (30S) subunits) and contain 23S (L), 16S (S), and 5S (L) rRNAs. Each subunit (L and S) contains about 30 proteins. Initiation factors involved are if1, if2, if3 and longation factors involved are ef-Tu, ef-Ts, and G. Translation is initiated with fmet (formylated Met). As many cp mRNAs contain a Shine-Dalgarno sequence preceding the first codon; it base-pairs with the 3'-end of 16S rRNA and translation is proceeded.In some cases Shine-Dalgarno sequence suppress the translation and proteins that bind the 5’ UTRs of mRNAs promote translation. Start codon is very important for starting translation at right codon. Internal ORFs are translated in to a polycistronic transcript.

Reference:-

Barkan A. (2011). Expression of Plastid Genes: Organelle-Specific Elaborations on a Prokaryotic Scaffold. Plant Physiology. 155: 1520–1532. Buchanan, B. B. Gruissem, W. Jones, L.R. 2005. Biochemistry & molecular biology of plants, 6th edition. Courier Companies, Inc., U.S.A, pp. 508- 565.

P.R. Archie. (2001). Rubisco. Encyclopedia Of Life sciences. 6: 125–149.

http://slideplayer.com/slide/6186650/.htm (2015.09.22).