An overview of chloroplast genomes Chloroplasts are autonomous genetic systems – they possess a complement of DNA that is expressed as protein Chloroplasts are descendants of ancient photosynthetic bacteria – the Endosymbiotic Theory Chloroplasts undergo a distinctive developmental program within the plant: Proplastid – an undifferentiated organelle present in meristematic cells Plastids – many different differentiated fates (chloroplast, leucoplast, …) The chloroplast is the differentiated state that is the most active in terms of gene expression Gene expression in the plastid is required for maintenance and differentiation For the most part (but not always), chloroplasts are inherited maternally Maternal (and paternal) inheritance is associated with cytoplasmically-inherited phenotypic traits: nuclear trait: NN x nn = nn x NN cytoplasmic C x c ≠ c x C trait: Plastid genome structure: 70-500 kbp (land plants 120-160 kbp) most chloroplast genomes contain an exact inverted repeat 10-1000 copies/plastid up to 50 plastids per cell -> as many as 5x104 copies per cell psaA psbD rbcL LSC chloroplast genome 5ʼ-rps12 120-180 kbp atpA IR IR 3ʼ-rps12 3ʼ-rps12 SSC 16S 16S 23S 23S ndhA The coding capacity of chloroplast genomes Chloroplast genomes encode three identifiable ribosomal RNAs 23S, 16S, 5S -> prokaryotic ribosomal RNAs; chloroplasts thus possess prokaryotic ribosomes, reflecting their evolutionary history Chloroplast genomes encode some 30 or so tRNAs (the number varies from species to species) This number of tRNAs is far fewer than the number of codons in the genetic code The complement of chloroplast protein-coding genes possesses all of the possible codons How do codons that are not explicitly specified by chloroplast-encoded tRNAs get translated by the ribosome? o “two out of three” – only the first two bases of the codon pair with the anticodon in the tRNA o “super-wobble” or extended wobble – the first base of the anticodon (complementary to the third base of the codon) is edited so that the modified base can pair with all four canonical bases o these mechanisms “expand” the decoding capacity of a small complement of tRNAs (tRNA transport is seen in mitochondria but not likely in chloroplasts) How many proteins are encoded by the chloroplast genome? Ribosomal proteins – 15-20 (note that the ribosomes have a full complement of >50; thus, most chloroplast ribosomal proteins are encoded by the nuclear genome) PEP-type RNA polymerase (all core subunits) 30-35 proteins involved in photosynthesis and carbon fixation (rbcL, subunits of PSI, PSII, ATP synthase, cytochrome b/f complex, and NADH dehydrogenase); all of these complexes possess nucleus-encoded subunits as well Intermediary metabolism (accD, chlI) Protein quality control (clpC, dnaK, groEL) Several as yet unassigned open reading frames Highly-purified Arabidopsis chloroplasts possess >1300 proteins identifiable by mass spectroscopy Chloroplast genomes encode <100 proteins most of the complement of chloroplast proteins are encoded by the nuclear genome protein import plays a large role in defining the chloroplast The biochemical complexes and pathways of the chloroplast are mosaics that consist of nucleus- and chloroplast- encoded subunits An overview of chloroplast transcriptional and posttranscriptional mechanisms Annual Reviews Transcription: Prokaryotic promoters and enzymes Two distinct DNA-dependent RNA polymerases PEP – analogous to the canonical bacterial multi-subunit DNA-dependent RNA polymerase (rpo), requires sigma factors for function Land plants possess several distinct sigma factors, probably with somewhat different functions or roles Sigma factors are nucleus-encoded NEP – single-subunit phage-type DNA- dependent RNA polymerase (T7 RNAP-like), nucleus-encoded RNA polymerases in the chloroplasts Chloroplasts have two different DNA-dependent RNA polymerases: E. coli-like (α, β, β' subunits, uses σ factors), chloroplast-encoded [PEP] phage (T7 RNA polymerase)-type, single subunit, nucleus-encoded [NEP] RNA processing: Endo- and exo-nucleolytic processing of the primary transcripts The players – prokaryotic ribonucleases RNAse J RNAse E PNPase These nucleases have modest or no RNA sequence specificity Specificity is conferred by RNA structures or accessory gene-specific RNA binding proteins The results are a panoply of RNA isoforms RNA processing: Maturation, editing, and removal of introns The players – prokaryotic ribonucleases RNAse J PNPase PPR/TPR proteins (RNA-binding proteins that are usually specific for a given processing or maturation reaction) RNA editing can change the coding capacity of mRNAs Chloroplast genes may possess introns – RNA sequences that are removed from the final, mature mRNA Chloroplast introns are related to Group I and Group II self-splicing introns, and are thus disnct from nuclear spliceosome-dependent introns (Caveat – Group II introns retain some RNA structures that are seen in various of the snRNPs of the nuclear splicing complex; these similaries point to a conserved chemical mechanism for splicing of nuclear and chlorplast Group II introns) Group I and Group II introns have disncve and essenal 3-dimensional structures Chloroplast introns require other proteins for efficient splicing Chloroplast splicing “factors” are RNA-binding proteins that have been co-opted for splicing hp://en.wikipedia.org/wiki/Group_II_intron Typical Group I intron structure: Splicing mechanism hp://en.wikipedia.org/wiki/Group_I_cataly:c_intron Intron splicing in chloroplasts is mediated or facilitated by specific splicing factors These factors ARE NOT homologous in sequence or function to the components of the nuclear spliceosome Annual Reviews Plas%d RNA edi%ng - overview “deaminase” PPR domain RNA binding protein C cis-element cis-element: each eding site (30-40 in angiosperm plasd genomes) is associated with a cis element that recruits the eding apparatus there is not a single, master element or mof; instead, most sites are controlled by separate dedicated factors Ø Site-by-site control of RNA edi%ng and gene expression PPR domain RNA binding protein PPR domain-containing proteins mediate numerous RNA processing and eding reac%ons in plasds angiosperm genomes encode 400-600 PPR proteins PPR proteins are responsible for edi%ng site specificity “deaminase” catalyzes the C->U (or occasionally U->C) reac%on a mul-subunit complex that includes the catalyc site The results: Populations of monocistronic and polycistronic mRNAs These mRNAs will have different 5’- and 3’- ends, as well as differing translatabilities and stabilities 3 types of chloroplast gene can exist in principle: • genes transcribed solely by PEP (class I) • genes transcribed by PEP and NEP (class II) • genes transcribed solely by NEP (class III) Class I Class II Class III psaA atpB accD psbB clpP rpl33/rps18 psbE ndhB ycf2 petB ndhF rpoB (?) ndhA rps16 rps14 rrn rbcL atpI psbA psbD Why two RNA polymerases? • PEP - functions in green tisues, developed chloroplasts • NEP - present in proplastids, functions to maintain proplastids (and perhaps contribute to alternate developmental fates of proplastids) Chloroplast gene transcripon during the proplasd->chloroplast transi8on PEP-dependent expression NEP-dependent Onset of germinaon mature chloroplasts § A switch from NEP-dependent to PEP-dependent transcrip8on occurs early during seedling growth § NEP-dependent transcripon remains relavely constant while PEP-dependent transcripon seems to be coupled to photosynthe8c capabilies Chloroplast gene expression in mature chloroplasts during a dark->light transi8on Mature plants grown in a normal light-dark cycle Shi1 to light Extended dark adaptaon measure PSI 1000 Measurements: 100 Prot. – immunoblot psa prot. Prot. Rate – pulse chase intact 10 psa prot. rate chloroplasts with labeled amino acids, psa mRNA SDS-PAGE, autoradiography 1 mRNA – northern blot psa transcr. Transcr. – pulse chase lysedchloroplasts 0.1 with labeled UTP, probe filters (as with 0.01 0 75 50 nuclear run-on assays) 25 125 100 hrs in light (data are taken from Klein and Mullet [Control of gene expression during higher plant chloroplast biogenesis, J. Biol. Chem. 262, 4341-4348, 1987] and Mullet and Klein, Transcrip,on and RNA stability are important determinants of higher plant chloroplast RNA levels, EMBO J. 6, 1571-1579, 1987) Chloroplast gene expression in mature chloroplasts during a dark->light transi8on Mature plants grown in a normal light-dark cycle Shi1 to light Extended dark adaptaon measure PSII 1000 Measurements: 100 Prot. – immunoblot psb prot. Prot. Rate – pulse chase intact psb prot. rate chloroplasts with labeled amino acids, 10 psb mRNA SDS-PAGE, autoradiography psb transcr. mRNA – northern blot 1 Transcr. – pulse chase lysedchloroplasts with labeled UTP, probe filters (as with 0.1 0 75 50 nuclear run-on assays) 25 125 100 hrs in light (data are taken from Klein and Mullet [Control of gene expression during higher plant chloroplast biogenesis, J. Biol. Chem. 262, 4341-4348, 1987] and Mullet and Klein, Transcrip,on and RNA stability are important determinants of higher plant chloroplast RNA levels, EMBO J. 6, 1571-1579, 1987) Chloroplast gene expression in mature chloroplasts during a dark->light transi8on Mature plants grown in a normal light-dark cycle Shi1 to light Extended dark adaptaon measure PSI PSII 1000 1000 100 100 psa prot. psb prot. 10 psa prot. rate psb prot. rate 10 psa mRNA psb mRNA 1 psa transcr. psb transcr. 1 0.1 0.1 0.01 0 50 25 75 0 100 125 50 75 25 125 100 hrs in light hrs in light