VL 1

semi autonomous organelle:

• own DNA

• own protein synythesis#proliferation by division

• energy metabolsim

• biosynthesis of

  • chlorophylls

  • carotenoids

  • fatty acids

  • purines, pyrimidines

• nitrite- and sulphate reduction

• circular, 40 - 50 µm

• freeof histones

• no 5-methylcytosin

• 150 kbp

• equals 1/30the genome of cyanobacteria, 1/1000 of nuclear genome

  • 10-50 plastomes/chloroplast

  • 50-200 chloroplast/cell

    =500-10000 plastomes/cell

  • 10% of the DNA of cells (bp)

ca. 120 genes

• • rRNA genes encode ribosomal RNA, which forms the structural and catalytic core of ribosomes.

  • tRNA genes provide transfer RNAs needed to read codons during translation.

  • Ribosomal protein genes encode some components of the chloroplast ribosome.

  • RNA polymerase subunit genes help build the plastid transcription machinery, especially the plastid-encoded polymerase system discussed later in the lecture.

  • Translation and elongation factors help protein synthesis proceed efficiently once mRNA is being read by ribosomes

• Subunits of protein complexes of the thylakoid membranes” means parts of big complexes like Photosystem I, Photosystem II, cytochrome b6f, or ATP synthase are still plastid encoded.

  • “LSU of RubisCO” means the large subunit of RubisCO is encoded in the chloroplast genome, while later slides show that the small subunit is encoded in the nucleus

• in single copy regions:

  • SSC = samll single copy regions

  • LSC: large single copy regions

• inverted repeats: IRa, IRb

• relic plastids with circular plastid-liek DNA with rRNA, tRNA, and protein genes, and they have functions that are medically relevant, which is why they are interesting drug targets

• medical use, since they produce iroprenoids and fatty acids

• polymerase

• promotors

• operons

• ribosomes

• introns

• editing

• two polymerases

  1. encoded in the plastid (PEP: plastid encoded RNA polymerase):

     transcription of proteins specific to the plastid

  2. nuclear encoded and imported (NEP: nuclear encoded RNA polymerase, homologous to bacteriophage polymerase): transcription of housekeeping genes

• plastid encoded RNA polymerase:

1. transcription of proteins specific to the plastid

• have specific promotors

nuclear encoded RNA polymerase

because the RNA sequence may need to be corrected before a functional protein can be made

• control of transcirption:

  • via transcription factors

    • encoded in nucleus and regulated light, hormones and other receptors

  • mostly encoded in the nucleus

  • activated via receptors (light, hormones,…)

• control translation:

  • by environmental factors

  • metabolic compounds

  • hormones (during development)

• regulation anterogade as well as retrogade:

  i.e nucleus influence plastid and vice versa

• shine dalgarno sequence is weak in plastids, the 5`UTR becomes decisive for translation control

• Secondary RNA structure and nuclear-encoded RNA-binding proteins are important here

• activation by a complex of nuclear encoded proteins

  > light : de-phosphoryltion - oxidation followed by reduction - translation

  > dark: phosphorylated - redox insensitive - no translation

• fine tuning lighht intensity

• addition of genes

• knock out via random insertion of resitance cassetes to create libraries of mutants

  > gene of interest probably knocked out, sometimes knocked-down oder even knocked-on, crossing is needed to gain homozygote plants!

• knocked out can be inserted by CrispR-Cas

  
  

• direct replacement of plastdidc genes is possible, because homologous recombination is the main mechansim of repair

• plastids are maternally inherited

  > no pollen distribution by GMOs

• 
  

• constraints because coding on both strands and operons

• only replacements of plastidic genes possible

• takes ages

• plastidic genome of the species in question has to be sequenced (because homologous recombination is used)

• method to regenrate callus has to exist

1. choose region of interest

1. biolistic transformation: preperation of DNA and creation of sterile pieces of leaves

1. several rounds of selevtion:

1. regeneration of the leaf pieces to plants (growing)

2. testing concerning homoplasticity via PCR

• herbicide resistance

• resistance against freezing

• resistance against pathogens

• production of secondary plant compunds (alkaloids) as pharmaceuticals

• production of proteins as pharmaceuticals (vaccines)

• addition of e.g. vitamins (Golden Rice)

• change of starch composition (increase of amylopectin in potato)

• research

• plants are able to form hybrids, i.e. foreign genes are spread via pollen: crops usually have ancestor that live in the same environment, and they still are able to cross (vertical gene transfer)

• release into the wild

• bacterial/virus can transmit genes: especially dangerous for antiobiotic resistance (horizontal gene transfer)

  
  

• bacterial/virus can transmit genes

• spreading of genes only possible via seeds

• release into the wild

• spreading of resistance:

  • different selection markers (sugar metabolism)

  • different methods for transformation (mico injection w/o selection markers)

• vertical spreading of genes:

  • transformation of plastids

• during transcription and translation

top transcription

bottom translation

• has to passage the envelope

• a sequence to recognize the proteins for transport is need for transport in the inside

• 
  

• by the tic/toc complex

• there are further transport systems concenring the envelope, also for envelope proteins themselves

• proteins of the thylakoid membrane or lumenal proteins have a thylakoid doimain in additoíon

• during protein folding:

  chlorophylls and carotenoids

• before folding:

  cytoctome: lumenal side, depending on protein

  Fe-S-centre: stromal side, under reductive conditions

Both came together with caperons!!

• LSU in the plastid

• SSU in the nucleus

• turnover of D1 protein: 30 min