Molecular Biology and Biotechnology WS23/24 Lecture Notes PDF

Molecular Biology and Biotechnology Institute for Molecular Biology and Biotechnology of Prokaryotes  Molecular Biology and Biotechnology WS23/24...

Molecular Biology and Biotechnology Institute for Molecular Biology and Biotechnology of Prokaryotes  Molecular Biology and Biotechnology WS23/24 Wednesday 8.30-10.00 lecture hall H15 Thursday: 8.30-10.00 lecture hall H13 The archaeal origins of the eukaryotic cell 25.10. Wednesday Genome Organization and Function 2.11. Thursday RNA Metabolism in Archaea and Eukarya 8.11. Wednesday written test: Protein Metabolism and Translation in Archaea and Eukarya 9.11. Wednesday 11.1.24 RNA Interference 15.11. Wednesday questions will RNA Editing 16.11. Thursday be in English CRISPR-Cas for defence and as a tool 29.11. Wednesday and German Rice Bacterial Blight mit CRISPR-Cas 7.12. Thursday Agricultural Applications of Genetically Modified Organisms?? 13.12. Wednesday Tutorial & Questions 20.12. Wednesday Written test 11.1. Thursday  Institute for Molecular Biology and Biotechnology of Prokaryotes CRISPR-Cas and RNA Metabolism in Archaea  we offer Master theses Small RNAs have different regulatory roles Topics: CRISPR-Cas small regulatory RNAs Ribonucleases Techniques used: genetics, molecular biology, microbiology, biochemistry (protein and RNA) (working with DNA, proteins and RNA)  Examination Board/ Prüfungsausschuss chairman for Biology: Marco Tschapka chairwoman for Biochemistry: Anita Marchfelder Information on extending deadlines etc. links: Biochemistry: https://www.uni-ulm.de/nawi/bio2/teaching/pruefungsausschuss-biochemie/ Biology: see Biology Homepage: https://www.uni- ulm.de/nawi/nawibiologie/fachbereich-biologie-startseite/ First semester Master lecture: (including: Biology Bachelor, Biochemistry Bachelor, from Ulm and other Universities) -> starts with some revision and summary of Bachelor knowledge The archaeal origins of the eukaryotic cell  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles up to 1977: two domains: Prokarya and Eukarya Carl Woese three domains: Bacteria, Archaea and Eukarya  An independent domain of life: Archaea -> three domains Only freaks living in extreme conditions? Archaea on Mars? Biotechnologists would love to grow all archaea for their enzymes, which withstand heat, acids, and salt. Biotechnology: thermophilic > 45 C (up to 113 C) psychrophilic < 20 C (below 0 C) acidophilic < pH 5 (below pH 1) alkalophilic > pH 9 (up to pH 11) halophilic up to 4 M KCl barophilic up to 40 MPa  Not only freaks but also important part of "normal" habitats! Archaea are everywhere! Archaea are similar to bacteria: … morphology … metabolism … genome organisation Archaea are even more similar to eukaryotes: … DNA replication … transcription … translation  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles R ES E A RC H | R E V IE W archaeal superphyla A B Euryarchaeota Euryarchaeota Crenarchaeota Nanoarchaeota 1990-2002 C Euryarchaeota Methanomicrobia Methanomassiliicoc. Verstraetearchaeo Geoa Methanocella Thermoplasmata MG II Korarchaeota Methanosarcina Aciduli- MBGD  indicates that B TACK versity may be Euryarchaeota Korarchaeota Furthermore Crenarchaeota Crenarchaeota of SA1 archaea methanogenic Thaumarchaeota branch basally Nanoarchaeota archaea, referr Aigarchaeota have a methyl genic lifestyle 90-2002 2002-2011 liicoccales (64) Previously u TACK superp TACK (Proteoarchaeota) Marine subsur Methanomassiliicoc. Verstraetearchaeota Geoarchaeota inated by mem moplasmata MG II Thermoproteales uli- MBGD Korarchaeota Desulfurococcales MCG), which ndi Methano- Sulfolobales archaeal clade Theion- fastidiosa Fervidicoccales  Nanoarchaeota Aigarchaeota hav gen 1990-2002 2002-2011 liico Pre C TAC Euryarchaeota TACK (Proteoarchaeota) Mar Methanomicrobia Methanomassiliicoc. Verstraetearchaeota Geoarchaeota inat Methanocella Thermoplasmata MG II Thermoproteales Aciduli- MBGD Korarchaeota Desulfurococcales MC Methanosarcina profundi Methano- Sulfolobales arch Haloarchaea Theion- fastidiosa Fervidicoccales arch. Hades-/ form ANME-1 (Fig MSBL Methano- archaea Thaumarchaeota gen Archaeoglobi bacteria/ pyri Thermococci this Methanococci Aigarchaeota boli Aenigmarchaeota DPANN Micrarchaeota Bathyarchaeota Asgard this Parvarchaeota Nanoarchaeota Altiarchaea Heimdallarchaeota pote Thorarchaeota arch Pacearchaeota Lokiarchaeota grou Woesearchaeota and Nanohalo- 2011-2017 Odinarchaeota archaeota Diapherotrites ous Fur Fig. 2. The expanding archaeal diversity. Schematic depiction of how the shape of the archaeal that tree of life has expanded over the years, revealing the major impact of cultivation-independent grow genomics pep DPANN: during the past 5Parvarchaeota, Diapherotrites, years. (A) Between 1990 and 2002,Nanohaloarchaeota, Aenigmarchaeota, only two archaeal phyla andwere known (24, (Euryarchaeota and Crenarchaeota). (B) Additional phyla were identified between 2002 and 2011, Nanoarchaeota may but the phylum-status of Nanoarchaeota was still controversial. (C) Since 2011, various additional Archaea today: four superphyla 1 2 3 4 Archaea come in squares!! Haloquadratum walsbyi Archaea are heat resistant! archaeal viruses are also special  General features of the Archaea Phenotypically, the Archaea are a lot like Bacteria. Most small (0.5-5 microns) rods, cocci, spirill, and filaments. The genomes of Archaea are generally 2-4 Mbp in size, similar to most Bacteria. One of the unique features: membrane lipids, which are ether (not ester) - linked. archaea bacteria archaeal membranes comprise isoprenen based lipids attached to glycerol-1-phosphate via ether bonds  Bacteria and Archaea Property Bacteria Archaea made of various cell wall made of peptidoglycan materials, not peptidoglycan fatty acids present, isoprenes present, linked lipids linked by ester bonds by ether bonds core: single large enzyme; RNA core: single small many subunits; similar to polymerase enzyme; 4 subunits eukaryotic RNA pol II protein 1st amino acid = 1st amino acid = synthesis formylmethionine methionine Even Woese long assumed that Archaea were but an ecological sideshow today. They seemed to live only in freak environments: in the middle of hot springs, in salt lakes like the Dead Sea, or in oxygen- starved swamps and to be few in both number and species. “They were confined, and there was a feeling that they couldn’t compete in aerobic conditions” says Woese. Archaea have been found far from the hot springs and swamps that were once thought to confine them. Ocean archaea are numerous. DeLong has discovered that nearly a third of the microbes in surface water off Antarctica are archaea. Fuhrman meanwhile has found signs that archaea are actually the dominant type of microbe in deep-ocean water. If you assume his samples from nine locations are representative of the whole deep ocean, says Fuhrman-- “there’s a very good chance that these are the most common organisms on Earth.” Many novel Archaea have been found in ”non- extreme” environments. The most interesting things about archaea may remain hidden, though, until researchers can examine the actual living organisms rather than their rRNA; although dead specimens have been isolated, the bugs have proved VERY hard to grow in culture. Biotechnologists would love to grow archaea for their enzymes, which withstand heat, acids, and salt. Model organisms: Haloferax volcanii, Methanocaldococcus, Methanosarcina, Thermococcus kodakarensis Sulfolobus solfataricus Model organisms: Haloferax volcanii, Methanocaldococcus, Methanosarcina Thermococcus kodakarensis Sulfolobus solfataricus easy and fast to grow easy to analyse genetic systems availabe "Omics" data (genome sequence, transcriptome, proteome) representative for a group of organisms  Euryarchaea All three phenotypes of Archaea are present in Euryarchaea: methanogens sulfur-dependent thermophiles: Thermococcus, Thermoplasma and Archaeoglobus extreme halophiles  Euryarchaea: Methanogens Key genera: Methanobacterium, Methanocaldococcus, Methanosarcina microbes that produce CH4 found in many diverse environments taxonomy based on phenotypic and phylogenetic features common organisms, found in all types of anaerobic environments: sediments and soils, animal guts, wastewater, oil deposits - natural gas is methane, produced by methanogenes  Euryarchaea: Methanogens Methanocaldococcus jannaschii Methanopyrus Methanopyrus Methanothermus Euryarchaea: hyperthermophilic Three phylogenetically related genera of hyperthermophilic Euryarchaeota: Thermococcus Pyrococcus Methanopyrus Euryarchaea: Haloarchaea Key genera: Halobacterium, Haloferax, Natronobacterium extremely halophilic Archaea have a requirement for high salt concentrations (typically require at least 1.5 M (~9%) NaCl for growth) found in artificial saline habitats (e.g., salted foods), solar salt evaporation ponds, and salt lakes model organism used: Haloferax volcanii easy to grow grows fast genome is sequenced (4 Mb) genetically tractable transcriptome- and proteome studies possible representative for the phylum Haloarchaea halophilic archaeon: requires high salt concentrations for growth important characteristics for a model organism  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles Recent development: back to only 2 domains BUT these new 2 domains are different from the ones before (only the number is the same). Eukaryotes evolved from Archaea 2015: sampling in the North Sea Lokis Castle: Loki-Archaeota 3.000 m below the see level, total darkness, high pressure, 300 C° Black smoker black smoker can be found around the world black smoker so far only cultured archaea investigated (restricts analyses) *new*: DNA is extracted from sampled probe (mixture of genomes!): metagenomes from these data genomes are assembled -> Lokiarchaeon https://astrobiomike.github.io/misc/amplicon_and_metagen ARTICLE doi:10.1038/nature14447 Complex archaea that bridge the gap between prokaryotes and eukaryotes Anja Spang1*, Jimmy H. Saw1*, Steffen L. Jørgensen2*, Katarzyna Zaremba-Niedzwiedzka1*, Joran Martijn1, Anders E. Lind1, Roel van Eijk1{, Christa Schleper2,3, Lionel Guy1,4 & Thijs J. G. Ettema1 The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of ‘Lokiarchaeota’, a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic ‘starter-kit’ to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes. Cellular life is currently classified into three domains: Bacteria, tant archaeal homologues of actin25 and tubulin26, archaeal cell division Archaea and Eukarya. Whereas the cytological properties of proteins related to the eukaryotic endosomal sorting complexes Bacteria and Archaea are relatively simple, eukaryotes are character- required for transport (ESCRT)-III complex27, and several informa- ized by a high degree of cellular complexity, which is hard to reconcile tion-processing proteins involved in transcription and translation2,17,23. given that most hypotheses assume a prokaryote-to-eukaryote trans- These findings suggest an archaeal ancestor of eukaryotes that might ition1,2. In this context, it seems particularly difficult to account for the have been more complex than the archaeal lineages identified thus suggested presence of the endomembrane system, the nuclear pores, far2,23,28. Yet, the absence of missing links in the prokaryote-to-eukaryote  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles new type of Archaea: Lokiarchaeon three domains closely related to eukaryotes, many proteins with high similarity to eukaryotic ones, e.g. proteins for phagocytosis two domains aus: Braun V. (2015) Biologie in unserer Zeit, 45: 212-213  What did they find: Many ESPs: 175 (3,3%) of the encoded proteins have high similarity to eukaryotic proteins. Examples for ESPs: actin: important for cytoskeleton, phagocytose (organelles!) small GTPases ESCRT protein complex (involved in membrane remodeling) ESP: eukaryotic signature protein, ESCRT: endosomal sorting complexes required for transport  Critique: Data are obtained from metagenomes! -> If the genome is not assembled correctly, the ESPs can come from contamination by eukaryotic DNA. Final proof required: growth of organism! ESP: eukaryotic signature protein https://astrobiomike.github.io/misc/amplicon_and_metagen  Many new metagenome data reveal: -> a lot of new Archaea R ES E A RC H | R E V IE W Asgard 3. A Archaea B Crenarchaeota Eukarya C TACK Eukarya Bacteria Eukarya 4. Bacteria Euryarchaeota Bacteria Euryarchaeota 5. DPANN 6. D Information processing Cell division/ cytoskeleton Endosomal sorting UB Trafficking machinery OST 7. so far only 2 Archaea clades: Lokiarchaeota Thorarchaeota Crenarchaea Heimdallarchaeota und Euryarchaea Odinarchaeota now additionally: Eukarya TACK, Asgard, DPANN 8. Thaumarchaeota Aigarchaeota 9. Bathyarchaeota Korarchaeota  long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon—‘Candidatus Prometheoarchaeum syntrophicum’ strain MK-D1—is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like All Asgard genomes from metagenomic studies! intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically Important: obtain an archaeon in culture. complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle–engulf–endogenize (also known as E3) model. How the first eukaryotic cell emerged remains unclear. Among vari- ous competing evolutionary models, the most widely accepted are Isolation of an Asgard archaeon symbiogenic models in which an archaeal host cell and an alphapro- Setting out to isolate uncultivated deep marine sediment microor- teobacterial endosymbiont merged to become the first eukaryotic ganisms, we engineered and operated a methane-fed continuous-flow cell1–4. Recent metagenomic characterization of deep-sea archaeal bioreactor system for more than 2,000 days to enrich such organisms group/marine benthic group-B (also known as Lokiarchaeota) and from anaerobic marine methane-seep sediments15 (Supplementary the Asgard archaea superphylum led to the theory that eukaryotes Note 1). We successfully enriched many phylogenetically diverse yet- originated from an archaeon that was closely related to these lin- to-be cultured microorganisms, including Asgard archaea members eages5,6. The genomes of Asgard archaea encode a repertoire of (Loki-, Heimdall- and Odinarchaeota)15. For further enrichment and proteins that are only found in Eukarya (eukaryotic signature pro- isolation, samples of the bioreactor community were inoculated in glass teins), including those involved in membrane trafficking, vesicle tubes with simple substrates and basal medium. After approximately formation and/or transportation, ubiquitin and cytoskeleton forma- one year, we found faint cell turbidity in a culture containing casamino tion6. Subsequent metagenomic studies have suggested that Asgard acids supplemented with four bacteria-suppressing antibiotics archaea have a wide variety of physiological properties, including (Supplementary Note 2) that was incubated at 20 °C. Clone library- hydrogen-dependent anaerobic autotrophy7, peptide or short-chain based small subunit (SSU) rRNA gene analysis revealed a simple com- hydrocarbon-dependent organotrophy8–12 and rhodopsin-based munity that contained Halodesulfovibrio and a small population of phototrophy13,14. However, no representative of the Asgard archaea Lokiarchaeota (Extended Data Table 1). In pursuit of this archaeon, has been cultivated and, thus, the physiology and cell biology of which we designated strain MK-D1, we repeated subcultures when this clade remains unclear. In an effort to close this knowledge gap, MK-D1 reached maximum cell densities as measured by quantita- we successfully isolated an archaeon of this clade, report its physi- tive PCR (qPCR). This approach gradually enriched the archaeon, ological and genomic characteristics, and propose a new model for which has an extremely slow growth rate and low cell yield (Fig. 1a). eukaryogenesis. The culture consistently had a 30–60-day lag phase and required more 1 Institute for Extra-cutting-edge Science and Technology Avant-garde Research (X-star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan. 2Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. 3Department of Civil and Environmental Engineering, Nagaoka University of Technology, Nagaoka, Japan. 4Kochi Institute for Core Sample Research, X-star, JAMSTEC, Nankoku, Japan. 5Biogeochemistry Program, Research Institute for Marine Resources Utilization, JAMSTEC, Yokosuka, Japan. 6Department of Marine and Earth Sciences, Marine Work Japan, Yokosuka, Japan. 7Research Institute for Global Change, JAMSTEC, Yokosuka, Japan. 8Research Institute for Marine Resources Utilization, JAMSTEC, Yokosuka, Japan. 9National Institute for Physiological Sciences, Okazaki, Japan. 10Section for Exploration of Life in Extreme Environments, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Japan. 11These authors contributed equally: Hiroyuki Imachi, Masaru K. Nobu. *e-mail: [email protected]; [email protected] Nature | Vol 577 | 23 January 2020 | 519 Candidatus ‘Prometheoarchaeum syntrophicum’, Masaru Nobu, a microbiologist at the National Institute of Advanced Industrial Science and Technology in Tokyo doubling time 1-2 weeks Amber Dance The mysterious microbes at the root of complex cells (2021) Nature 593: 328-330  genomic DNA from this culture confirms previous metagenome data metabolism verified predictions that were based solely on the metagenome analysis methods used for culturing will allow to cultivate more Asgard archaea The Asgard ESPs are enriched in proteins that are involved in membrane trafficking, vesicle formation and transport, cytoskeleton formation, and the ubiquitin network, which suggests that these archaea possess a eukaryote-type cytoskeleton and an intracellular membrane system. Asgard archaea have apparent evolutionary affinity with eukaryotes, confirmed by independent lines of evidence: (1) phylogenetic analysis of highly conserved genes and the detection of several ESPs that are absent or far less common in other archaea; (2) new metagenome data substantially extend the phylogenetic and functional diversity of the Asgard superphylum; (3) phylogenetic analyses of universally conserved genes from an expanded set of archaea, bacteria and eukaryotes support the two-domain tree topology, with the Heimdallarchaeota group being the most likely sister group of eukaryotes. UB: Ubiquitin OST: Oligosaccharyltransferase-Komplex Eme et al. "Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes " (2023) Nature, 618, pages 992–999 divides every 7-14 days scale bar 500 nm  Eukaryotes evolved from Asgard Archaea (1) genes for ESPs have been found in Asgard genomes (2) ESP proteins are involved in important eukaryotic functions like: membrane trafficking, vesicle formation and transport, cytoskeleton formation, and the ubiquitin network.  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles Where do organelles come from? Organelles 1. Endosymbiont and hydrogen hypotheses 2. Organellar genomes 3. Protein sorting 4. Organelle division  Endosymbiont hypothesis organelles derived from free living unicellular organisms ancestor of mitochondria: a-proteobacteria (proof: sequence analysis) ancestor of chloroplasts: cyanobacteria (proof: sequence analysis, they have chlorophyll a and not bacteriochlorophyll like other photosynthetic active bacteria) co-evolution with the host cell resulted in the evolution of organelles (from endosymbiont to organell) organelles contain: two membranes own DNA 70S ribosomes (similar to bacterial; eukaryote: 80S), reproduction by division (similar to bacteria) Original endosymbiont hypothesis: Lynn Margulis 1967 Endosymbiont hypothesis: 1. origin of the nucleus: karyogenetic hypothesis 2. uptake of a bacterium Advantage upon uptake of a bacterial cell: O2 - utilisation. Problems with the endosymbiont hypothesis: Host is a primitive eukaryote. Is the host a bacterium or an archaeon? -> We know now, it was an archaeon! Anaerobic, fermenting ur-eukaryote takes up a bacterium, which is only facultatively aerobic. Respiration at first not important (no O2). Why was it then taken up? Hydrogen hypothesis: (1998 Müller and Martin) Basis is the endosymbiont hypothesis. -> New part: symbiosis is forced! Anaerobic, strictly hydrogen dependent archaeon takes up a facultatively anaerobic, respiration-competent bacterium, bacterium evolves to mitochondrion, however it supplies at first H2 (not CO2). Bacterium Archaeon Hydrogen hypothesis -> confirms hydrogen hypothesis Asgard archaea have a lot of eukaryotic properties Amber Dance The mysterious microbes at the root of complex cells (2021) Nature 593: 328-330 Amber Dance The mysterious microbes at the root of complex cells (2021) Nature 593: 328-330 Candidatus ‘Prometheoarchaeum syntrophicum’, Masaru Nobu, a microbiologist at the National Institute of Advanced Industrial Science and Technology in Tokyo doubling time 1-2 weeks Amber Dance The mysterious microbes at the root of complex cells (2021) Nature 593: 328-330 Eukaryotes evolved from Archaea single (monophyletic) origin for endosymbiosis primary endosymbiosis: origin of mitochondria and chloroplasts a) archaeon + bacterium = eukaryote with mitochondrium b) eukaryote with mitochondrium + bacterium = eukaryote with mitochondrium and chloroplast secondary endosymbiosis: uptake of a eukaryotes (which has undergone primary endosymbiosis) by eukaryotes eukaryote + eukaryote = eukaryote mitochondria structure: - outer mitochondrial membrane - inner mitochondrial membrane cristae, many folds to enlarge the surface area (respiratory chain is located there, larger area: more ATP) - intermembrane space - matrix - 100 - 2,000 mitos/cell  initially one function: production of energy through respiration (ATP production) nowadays: involved in numerous metabolic processes: biosynthesis of amino acids, lipids, heme, and Fe-S clusters crucial functions in cellular signaling pathways, quality control, and programmed cell death defects of mitochondria lead to severe diseases of the nervous system, heart, muscles, and other tissues mitochondria from different eukaryotes show a lot of differences chloroplasts photosynthesis, storage (e.g. of starch), synthesis of fatty acids, terpene etc. comparison plastids-mitochondria: both are semi-autonomous, own DNA, two membranes, similar structures, own transcription- and translation apparatus mitochondria are smaller than chloroplasts Organelles 1. Endosymbiont hypothesis 2. Organellar genomes 3. Protein sorting 4. Organelle division mitochondrial genomes Organism Genome Size (kb) free living bacteria Escherichia coli 4,639 Rickettsia prowazekii 1,111 mitochondria Homo sapiens 16 Reclinomonas americana (protist) 69 plant mitochondria Marchantia polymorpha 186 Arabidopsis thaliana 267 Cucurbita pepo (pumpkin) 982 Citrullus lanatus (water melon) 2,400 Silene conica (striped corn catchfly) 11,000  genome sizes smallest genome: human mitochondrial genome 13 proteins are encoded only very small part of the proteins of a proteo- bacterium -> the rest of the protein genes have been transferred to the nucleus! high rate of mutations (not very well protected, repair mechanisms not very efficient)  Size trends in evolution of mtDNA o not a single trend o shrank in some lines, then got large again in others fungi à man: economization green algae à higher plants: enlargement  genomes from plant mitochondria are larger than from other organisms they contain introns they contain noncoding sequences genes are derived from plastids, nuclear and viral DNA (and unknown sources), some of these genes are transcribed, but it is not likely that they function  Plant mitochondrial DNA 1. size and arrangement highly variable 2. probably mostly linear ( human and yeast mt DNA) 3. no histones 4. low copy number per organelle (2-10 copies/ mt) 5. inherited uniparentally (with exceptions) (-> inheritance mostly maternally) DNA replication in human mitochondria (circular genomes) chloroplasts Genome Size free living bacteria Escherichia coli 4,63 Mbp Rickettsia prowazekii 1,11 Mbp Synechocystis 3,57 Mbp chloroplasts chloroplast genomes average 130 kb (range: 70 – 400 kb) genome typically comprises four segments: large region of single copy genes inverted repeat B inverted repeat A small region of single copy genes  multiple, related circles: 1 master circle and several subgenomic circles subgenomic circles derive from master circle by recombination at direct repeats more repeats, many more subcircles replication replication independent of replication in nucleus replication initiates in IR regions most of the proteins for replication are encoded in the nucleus replication higher plant chloroplasts: app. 80-120 proteins encoded in plastidial genome only approx. 5% proteins in comparison to the cyanobacterial genome -> the rest has been transferred to the nucleus altogether app. 2,500-3,000 proteins present in chloroplasts app. 100 chloroplasts per cell many copies of cp DNA / cp : 10-100 (varies widely with the developmental stage and type of plastid) operon organisation: often polycistronic transfer of DNA to the nucleus: (example: chloroplast) Anabaena variabiis Nostoc sp. genome size: 7.1 Mb protein ORFs: 5.661 Transfer of DNA to the nucleus Integration of DNA into the nuclear DNA Targeting of proteins to the organelle  Transfer of DNA to the nucleus Why? Complex gene regulation easier if genes are in the nucleus, regulation of general whole cell gene expression easier? Mitos: redox-associated reactions (lots of free radicals), danger to mitochondrial DNA (mutations). Selection for genes retained in organelles? 1. ribosome-subunits (RNA and proteins) 2. structural proteins, which are involved in maintaining the redox balance via the bioenergetic membrane the remainder of proteins is nuclear encoded -> requires import into organelles Organelles 1. Endosymbiont hypothesis 2. Organellar genomes 3. Protein sorting 4. Organelle division transport through nuclear pores transport through membranes transport in vesicles Two types of cytosolic ribosomes: free and membrane-bound They synthesize proteins with different destinations. 1. Free ribosomes 2. membrane bound ribosomes: Peptide sequences for targeting to different organelles type of organelle targeting domain ER signal peptide (SP) Nucleus nuclear localization signal (NLS)(internal sequences, positively charged aa: Lys, Arg) Peroxisome peroxisomal targeting signal(s) (PTS1 and PTS2) Vacuole vacuolar sorting signal (VSS) Chloroplast transit peptide (TP), N-terminal Mitochondrion pre-sequence, N-terminal  Targeting of proteins destined for chloroplasts and mitochondria How are proteins targeted to chloroplasts and mitochondria from the cytoplasm? How do they get through the membranes? import: chloroplast  import: chloroplast äCM: outer membrane: doesn´t need tp; IM: inter membrane space ; S: stroma; iCM: inner membrane; TM: thylakoidm.; L: lumen; for TM und L two signals required: 1. tp into cp ; 2. hydrophobic signal for transport into lumen or thylakoid membrane  = transit peptide Import: protein biosynthesis in the cytosol kept unfolded by chaperons binding of the pre-protein to the receptor transport through the membranes removal of transit peptide inside the organell: folding of the protein integration into protein complex Chaperones play roles in membrane transport on both sides of the membrane. Features of chloroplast protein import 1. post-translational 2. proteins synthesized as precursors with an amino(N)-terminal extension: transit peptide 3. transit peptide acts as the “zip code”, removed during or soon after import 4. Different chaperones bind to precursor before, during and after membrane translocation. Hsp70-type chaperones maintain partially folded state in cytoplasm, whereas Hsp60 (cpn60) and Hsp70 promote folding inside organelle. 5. ATP and GTP are also required for envelope membrane translocation.  6. Import receptors and translocation complexes (i.e., Tocs and Tics) assemble at envelope membrane contact sites. – proteins of the outer membrane complex are called Tocs – inner membrane translocon complex proteins are called Tics 7. After import, specific endoproteases in stroma remove transit peptide sequences. Toc: translocase of the outer chloroplast membrane, Tic: translocase of the chloroplast inner membrane  Toc Toc: translocase of the outer chloroplast membrane Tic Tic: translocase of the chloroplast inner membrane Fig. 4.6, Buchanan et al. Targeting to inner chloroplast compartments: thylakoid membrane- spanning and lumen proteins Proteins destined to the inner compartment (i.e., thylakoid-membrane spanning and lumen proteins) have longer transit peptides with 2 zip codes. They are removed in two steps: cleave cleave Precursor à Intermediate à Mature  the first cleavage unmasks a second sorting signal (zip code) the intermediate goes to the inner compartment the second cleavage generates the mature protein  Toc Tic chanan et al.  Import into mitochondria: pre-sequence, N-terminal, a-helix with positively charged aa, non- polar aa (amphi-philic helix) amphiphilic helix: hydrophobic amino acids: Ala, Leu almost entirely on one site of the helix; charged amino acids (Arg) on the other  Mitochondrial import complexes pre-sequence TOM: translocase outer mitochondrial membrane; TIM: translocase inner mitochondrial membrane; mtHsp70 pulls protein into matrix different destinations: different import pathways -> matrix -> inner membrane intermembrane space mRNA -> protein z.B.: minsmpylhknlrllrllssksspfplslrpfsprsfslstlfssssssssmenneatngsksssnsfvfnkrraegfditdkkkrnlerksq klnptntiayaqilgtgmdtqdtsssvllffdkqrfifnageglqrfctehkiklskidhvflsrvcsetagglpgllltlagigeeglsvnv wgpsdlnylvdamksfipraamvhtrsfgpsstpdpivlvndevvkisaiilkpchseedsgnksgdlsvvyvcelpeilgkfdlekakkvfg vkpgpkysrlqsgesvksderditvhpsdvmgpslpgpivllvdcpteshaaelfslkslesyysspdeqtigakfvnciihlspssvtsspt yqswmkkfhltqhilaghqrknmafpilkassriaarlnylcpqffpapgfwpsqltdnsiidptpsnkcsssnlaesisaenllkfnlrpva irgidrscipapltssevvdellseipeikdkseeikqfwnkqhnktiieklwlsecntvlpnclekirrddmeivilgtgssqpskyrnvsa ifidlfsrgsllldcgegtlgqlkrrygldgadeavrklrciwishihadhhtglarilalrskllkgvthepvivvgprplkrfldayqrle dldmefldcrsttatswaslesggeaegslftqgspmqsvfkrsdismdnssvllclknlkkvlseiglndlisfpvvhcpqaygvvikaaer vnsvgeqilgwkmvysgdsrpcpetveasrdatiliheatfedalieealaknhsttkeaidvgsaanvyrivlthfsqrypkipvideshmh ntciafdlmsinmadlhvlpkvlpyfktlfrdemvededaddvamddlkeeal protein will be in the nucleus, the cytosol, plastids, mitochondria....? (problem in plant cells: mitochondria and plastids have similar pre-sequences, import machines) Approaches: 1. In silico 2. Experimental in vitro-import-assays GFP-fusion proteins Bioinformatic predictions of subcellular location for both mt and cp: several arginines, serines, leucines, few or no acidic amino acids mitochondria: amphiphilic helices with one side positively charged (within the first 10-15 amino acids) and one hydrophobic side, app. 20- 60 amino acids long plastids: app. 20-100 amino acids long Bioinformatic predictions of subcellular location Prediction programmes: Target P – predicts whether the protein is targeted to chloroplasts, mitochondria, cytosolic or secreted ChloroP – predicts whether the protein is targeted to chloroplasts Psort - predicts whether the protein is targeted to chloroplasts, mitochondria or cytosolic Predotar – predicts whether the protein is targeted to chloroplasts, mitochondria or the ER Experimental approaches 1. in vitro import assay Transport into organelles can be carried out in cell- free systems using in vitro- synthesized precursors. Amino acids: cysteine and methionine (usually used methionine) In vitro-import: incubation of proteins with isolated organelles recombinant protein incubate with isolated organelles (radioactively labelled) transit peptide transit peptide removed! precursor- protein cleaved protein 1: before import 2: after import w/o protease 3. after import with proteas 2. GFP-fusion proteins pre-sequence or target peptide GFP-protein protoplast transformed with plasmid signal sequence cleaved off after import into cp and mt AthTrzL2- GFP filter to detect GFP filter to detect dye (and autofluorescence for mitos of chloroplasts) (mito tracker) AthTrzS2- RFP filter to detect filter to detect RFP autofluroescence of chloroplasts RNA import into organelles  RNA import into organelles definitely tRNAs, 5S rRNA (human) miRNAs other?? miRNA import: Organelles 1. Endosymbiont hypothesis 2. Organellar genomes 3. Protein sorting 4. Organelle division Organelles reproduce by divison. Similar to bacterial division, protein involved: FtsZ (filamentous ts), division is independent from cell cycle. Fusion and Fission: Fusion and Fission: plant chloroplasts: FtsZ Antisense plants (Arabidopsis) wt antisense gegen FtsZ mRNA Fusion and Fission: FtsZ1 FtsZ2 GFP-fusion proteins for localisation not only fission but also fusion turnover of damaged organelles Fusion and Fission:  the tree of life: from 2 domains to 3 domains introducing Archaea as new domain the tree of life: from 3 domains to 2 domains the ancestor of the eukaryotic cell organelles Questions?