Molecular Biology Test-Test-Script PDF

8 Molecular biology 8.1 Fachbegriffe für Molekularbiologie Ergänzen Sie die folgende Liste mit der deutschen Übersetzung. Falls Sie einen Begriff nicht kennen, lohnt es sich ein Glossar zu erstellen. Notieren Sie dabei den Begriff (D & E) und dessen Definition. Sie können sich daraus auch Karteikärt...

8 Molecular biology 8.1 Fachbegriffe für Molekularbiologie Ergänzen Sie die folgende Liste mit der deutschen Übersetzung. Falls Sie einen Begriff nicht kennen, lohnt es sich ein Glossar zu erstellen. Notieren Sie dabei den Begriff (D & E) und dessen Definition. Sie können sich daraus auch Karteikärtchen machen (auch digitale). Der Glossar ist eine gute Hilfe beim Lernen. Dasselbe gilt für die Fachbegriffe aller Themen. DNA strand molecule pathogenic, pathogenous chemical substance (to) transform (transformed, transformed) (culture) medium (plural: media) protein synthesis nucleic acid double helix nucleotide base pairing pyrimidine purine adenine guanine cytosine thymine uracil 5 pentose ribose deoxyribose phosphoric acid nucleoside adenosine adenosine triphosphate (ATP) deoxycytidine coenzyme phosphodiester bond 3’-end / 5’-end (“3 / 5 prime end”) genetic information RNA, ribonucleic acid deoxyribonucleic acid hydrogen bonds / bridges rope ladder coding = template /matrix / antisense strand replication transcription translation complementary strand backbone 6 gel electrophoresis single-stranded replication fork helicase single strand binding proteins DNA polymerase I / III ligase leading strand lagging strand Okazaki fragments primer primase telomeres gen, genes molecular life / biological clock germ-line cells telomerase amino acid protein chain mutation proofreading polymerase chain reaction amplification 7 denaturation annealing, hybridisation polymerisation / elongation / extending enzyme metabolism messenger RNA transfer RNA ribosomal RNA snRNA (small nuclear) siRNA (small interfering) copy ribosome amino acid chain clover-leaf spliceosome RNA interference to silence (silenced, silenced) promoter terminator initiation elongation termination 8 to synthesise (synthesised, synthesised) seamlessly coding introns exons alternative splicing RNA processing capping polyadenylation codon / triplet histidine glutamic acid methionine degenerated universal genetic code start signal / sequence stop sequence three-dimensional structure anticodon Aminoacyl tRNA synthetases active centre 9 genome mutation chromosome mutation gene mutation point mutation substitution silent mutation missense mutation nonsense mutation frame-shift mutation DNA damages lung cells UV light X-ray radiation cigarette skin cell repair enzymes yeast dimers spontaneous mutation deamination mutagens cross-linking stretching of the DNA 10 intercalating substances antibiotics cytostatics excision repair base analogues single strand breaks loss of end piece deletion insertion shift in the reading frame / reading-frame shift compartmentalisation gene expression destructive / catabolic processes constructive / anabolic processes operon substrate induction end product repression repressor binding site induction structural genes constitutive genes 11 regulated genes lactose (milk sugar) regulator gene tryptophan gene / genetic technology / genetic engineering gene therapy isolation recombination gene transfer selection cell reproduction / cell propagation restriction enzymes recognition sequence / site palindrome ( / inverted repeat) sticky ends blunt ends ori (= origin of replication) recombinant plasmid vector marker genes GFP ( = green fluorescent protein) microinjection 12 Agrobacterium calcium phosphate precipitation lipid infection electroporation particle gun genomic library hybridisation probe insulin genetic fingerprint microsatellites (= Short Tandem Repeats) transgenic / gene-modified gene pharming green gene technology Bt maize (Bt = Bacillus thuringiensis) Amflora potato antisense foreign gene 8.2 Historical experiments relating to the function of DNA In the first half of the 20th century, there was uncertainty as to which molecules in the cell stored the genetic information. The most likely "candidates" on offer were proteins and DNA. In 1928, Frederick Griffith showed that harmless bacteria become pathogenic if dead pathogenic (krankheitsauslösende) cells are added to the medium. The property (Eigenschaft) is transmitted to all offspring. There was obviously a chemical substance in the dead pathogenic bacteria that produced this inheritable change. The chemical nature of this substance, however, was unknown (book p. 120). 13 It was not until 1944 that Oswald T. Avery was able to show, by adding all the possible substances from pathogenic bacteria to the culture media of live, non-pathogenic bacteria, that this "transforming agent" (transformierende Prinzip) was DNA (book p. 120). In 1952, Alfred Hershey and Martha Chase showed that in viruses too, it was not proteins but nucleic acids that constituted the genetic material (book p. 175). There now began a competition between several research groups to decipher the three- dimensional structure of DNA. Two relatively unknown young scientists, James Watson and Francis Crick, were able to show in 1953 that DNA is composed of two strands (Stränge). This led to the now familiar concept of the DNA double helix. 8.2.1 Summary 14 8.3 Structure of the nucleic acids (Nukleinsäuren) DNA and RNA 8.3.1 Nucleotides (Nucleotide) They are the building blocks (Bausteine) of nucleic acids. (book p. 121/122). For their part, however, nucleotides consist of three subunits (Untereinheiten): One of the five bases (Basen): adenine, guanine, cytosine, thymine or uracil A pentose (sugar), either ribose or deoxyribose (Desoxyribose) A molecule of phosphoric acid (Phosphorsäure) Study the corresponding formulas in the Chemistry & in the Biology book (p. 121/122) and note the following details: Exact formula of phosphoric acid Difference between ribose and deoxyribose Purine and pyrimidine bases: recognise formulas Linkage of the elements to a nucleoside or nucleotide Acid Sugar Bases Nucleosides Nucleotides RNA Phosphoric acid Ribose Adenine Adenosine (A) Adenosine monophosphate (AMP) Guanine Guanosine (G) Guanosine monophosphate (GMP) Cytosine Cytidine (C) Cytidine monophosphate (CMP) Uracil Uridine (U) Uridine monophosphate (UMP) DNA Phosphoric acid Deoxyribose Adenine Deoxyadenosine (dA) dAMP Guanine Deoxyguanosine (dG) dGMP Cytosine Deoxycytidine (dC) dCMP Thymine Deoxythymidine (dT) dTMP Note: the energy-rich compound ATP is also derived from a nucleotide. In addition, nucleotides are part of the structure of the coenzymes NADH and NADPH (see chapter Metabolism / Stoffwechsel). Cf. formulas in your books. 15 8.3.1.1 Glossar zum Bau von DNA & RNA (Buch S. 121) DNS DNA Desoxyribose Deoxyribose Phosphorsäure phosphoric acid Basen / bases Pyrimidinbasen / pyrimidine bases Purinbasen / purine bases Nucleotid / nucleotide Nucleosid / nucleoside DNase 3’-Ende 5’-Ende Phosphodiesterbindung phosphodiester bond RNS RNA Ribose Uracil 16 8.3.2 Linking (Verknüpfung) of nucleotides Since phosphoric acid can form two ester bonds (phosphodiester bond, Phosphordiesterbindung), whole chains (Ketten) of nucleotides are possible. This involves the esterification of pentose with phosphoric acid at C3’ and C5’. Note: - At the beginning of the chain, the 3’-OH group of the first pentose is free. At the end of the chain, the 5’-OH group of the last pentose is esterified with phosphoric acid. The chain accordingly has one 3’-OH end and one 5’-P end. - In the synthesis of nucleotide chains, the energy-rich nucleoside triphosphates ATP, GTP, CTP, TTP and UTP are used as building blocks. With each extension of the chain by one nucleotide, diphosphate is cleaved off (abgespalten) in the reaction. - By international convention, the nucleotide sequences of the chains are read in the direction 5’ - 3’, using the one-letter code of the nucleotides (A, T, G, C, U). 8.3.3 DNA and RNA DNA is the carrier (Träger) of hereditary information (Erbinformation), while several different sorts of RNA fulfil important functions in protein synthesis. The chemical structure exhibits certain similarities: both are chains of nucleotides. The following differences may be highlighted: 17  DNA RNA Pentose Bases Number of strands Function Chemical analyses of DNA reveal a striking regularity: whatever the origin of the DNA, i.e. from bacteria to mammals, the proportions of A and T always match exactly and likewise those of G and C (Chargaff’s rule). The reason for this lies in the fact that DNA always occurs as a double molecule. The chemical structure of the four bases allows hydrogen bridges (Wasserstoffbrücken) between A and T and between G and C, but not "crosswise". These H bridges stabilise the spatial structure of the two DNA strands in the famous double helix, which can be pictured as a rope ladder (Strickleiter) twisted about itself: sugars and phosphate molecules form the two "ropes" (“Seile”) and two paired bases form a "rung" („Sprosse“). Only one of the two strands contains the correct information for the formation of mRNA (codogenic / template strand, codogener Strang), the other strand serves as a matrix for DNA replication (as well as the first discussed strand). Linking of the nucleotides occurs from the C atom 5’ of the deoxyribose via phosphoric acid to the C atom 3’ of the next deoxyribose. The bonds in the two strands run in the opposite direction: in one strand in the 3’ - 5’ direction, in the complementary strand in the 5’ - 3’ direction. Due to the many negatively charged phosphate groups that together with deoxyribose form the backbone (Rückgrat), DNA is negatively charged (geladen) and migrates to the positive pole (Anode) in gel electrophoresis. 18 The principle of base pairing (Basenpaarung) is the basis for many molecular biological processes: - DNA replication (chapter. 8.5): one strand serves as a template (Vorlage) for the synthesis of a second strand - Transcription (chapter.8.8) - Protein synthesis (chapter. 8.10) - Numerous in vitro analytical and synthetic procedures in molecular biology and genetic engineering laboratories (like PCR, chapter 8.6). RNA usually occurs as a single strand, which is related to its function (Chapter. 8.7). 8.4 Structure of chromosomes Biologiebuch S. 106 & Kapitel „Genetik“ im Skript des 1. Lehrjahres. During early interphase, each chromosome consists of only one chromatid and therefore of one DNA double helix. However, the DNA is not freely present but coiled around spherical proteins, histones (string of pearls structure = Perlenkettenstruktur). This string of pearls is again wound about itself and thus forms a chromatin fibre (Chromatinfaser). The chromatin fibre is about 15 times shorter than the DNA it contains. Before mitosis or meiosis, the chromatin fibre is first duplicated identically so that one chromosome now consists of two chromatids connected at the centromere. The chromatids now twist and fold further until they are maximally shortened and thickened in metaphase. A metaphase chromosome is about 7,300 times shorter than the DNA double strand it contains! Gen-Länge durchschnittlich 3'000 Basenpaare max. 2.4 Mio. Basenpaare Promotor-Länge ca. 100 Basenpaare Transkriptionsdauer theoretisch 50 sec, gibt aber Pausen Genom des Menschen 3.3 Mia. Basenpaare etwa 30'000 – 35'000 Gene nur 2 % codierend ca. 99.9% identisch zw. Individuen ca. 0.1 % unterschiedlich → 3 Mio. variable Stellen 19 8.5 DNA replication It can now readily be imagined how DNA is replicated (duplicated) in the S phase before cell division: The enzyme topoisomerase unwinds the DNA strand. With the aid of the enzyme helicase, the two DNA strands are separated from one another, held apart by strand-binding (SSB = single-strand binding) proteins and each made up to a double helix by free nucleotides binding to the open sites. The enzyme DNA polymerase III is responsible for this. Each of the new DNA double helices therefore contains a strand of the original double helix and a complementary copy. They are therefore identical (book p. 124). Since it is always an old & a new strand combined one speaks of “semi-conservative replication”. This could be proven in 1958 by the two biochemists Meselson and Stahl (book p. 123). The DNA strand is opened simultaneously at many sites so that replication can proceed synchronously. At the end, the pieces are linked by the enzyme DNA ligase to form continuous strands. DNA polymerase III can attach a further nucleotide only to a free 3’ end. Replication of the strands must therefore occur in the opposite direction, as can be seen in the Biology book on p. 124. One strand (leading strand, Leitstrang) can accordingly be synthesised continuously, while the complementary strand (lagging strand, Folgestrang) is formed piecewise "backwards". These initially not yet bound individual pieces are known as Okazaki fragments after their discoverer. The nucleotides are used in the form of triphosphates (ATP, TTP, GTP, CTP). Cleavage of P provides energy for synthesis. In addition, DNA polymerase III can only work if a start has already been made ( primer). Primers consist of RNA and are produced by the enzyme primase. At the end, the enzyme DNA polymerase I replaces the primers with DNA. The individual pieces are linked by the enzyme ligase. DNA replication is an unbelievably rapid process: the DNA of the largest chromosome of the fruit fly contains 62 million base pairs and is duplicated within 3 minutes! 20 A particular problem arises with replication at the ends of the chromosomes, known as telomeres (Telomere): the polymerase can only attach nucleotides to the 3’ end of an already existing strand (hence the primers). At the 5’ ends of the leading strand and the lagging strand, gaps remain after removal of the RNA primers that cannot be filled by polymerase. With each replication, therefore, the chromosomes become one piece shorter. So that no genes are lost, chromosomes have non-coding pieces at the ends, known as telomeres. They consist of 100 to 1,000 repetitions of the sequence TTAGGG. On replication in the S phase before each cell division, the chromosomes become one primer length shorter until the telomeres have broken down. Genes are then gradually lost so that the cell dies. In fact, cells can only divide about 30 to 50 times; accordingly the telomeres are a sort of hand on our molecular life clock, which shows the cell when its time has run out. Germ-line cells (egg cells and sperm) are the only cells that can divide as often as they “want” (otherwise life on earth would have become extinct long ago!). They possess an enzyme called telomerase that can constantly prolong the telomeres. Would we be immortal if all cells were to contain telomerase? Consider the accuracy with which the cell needs to control DNA replication! A single incorrect nucleotide can result later in the incorporation of an incorrect amino acid (Aminosäure) and hence in a non-functional protein. A break in the protein chain is also possible. Mutations can therefore arise from errors during replication. However, cells possess repair systems: - The DNA polymerase III of bacteria checks its own work: whenever it has prolonged the strand by one base, it "goes back" and "reads" what it has "written" (proofreading), excises the incorrect nucleotides and replaces them with the correct ones. In eukaryotes, further proteins are added to the proofreading function of polymerase. - DNA that is not precisely replicated is also permanently exposed to many harmful influences and undergoes chemical changes (chapter 8.11). Fortunately, most damage is corrected in that each cell constantly checks and repairs its genetic material. More than 50 different types of DNA repair enzymes have been identified to date in higher organisms! However, it should not be forgotten that, while they are harmful in most cases, mutations are the driving force of evolution. Positive mutations made evolution possible in the first place! It is therefore not necessarily desirable for all changes in DNA to be completely corrected. 21 8.5.1 Hemmstoffe der DNA-Replikation Substanzklasse Substanz Wirkungsmechanismus Antibiotika hemmt Topoisomerase II & IV ( = Gyrase-Hemmer Gyrase) → verhindern Schliessen der (Verbindungen, die von 4- geöffneten Stränge und damit das z.B. Ciprofloxacin Oxocholin-3-carbonsäure Supercoiling → RNA expandiert bis Zelle abstammen) platzt Nur bei Prokaryonten Zytostatika Bindet an DNA (zwischen G und C) und „verklebt“ die Doppelstränge Komplexbildung mit den Guaninresten Replikationshemmer der DNA Actinomycin D (interkalierend) bei Pro- und Eukaryonten hemmt in niedrigen Konz. auch Transkription antibakteriell, aber zu toxisch Bildung kovalenter Quer-vernetzungen Replikationshemmer → verhindert Trennung der Mitomycin (interkalierend) Doppelstränge bei Pro- und Eukaryonten Alkylierung der DNA-Stränge → Replikationshemmer Strangbrüche & Vernetzung der DNA- Cyclophosphamid (interkalierend) Stränge & DNA-Protein-Vernetzungen hemmt auch Transkription Können als falsche Nukleotide nicht Nukleotidanaloga z.B. Cytosinarabinosid eingebaut werden und hemmen so die Polymerase 8.6 PCR PCR (polymerase chain reaction) is DNA replication in vitro. Read up on the principle of the procedure in the Biology book p. 126 and answer the following questions: - Make a list of the replication enzymes of the cell and consider which enzymes or components are necessary for PCR and which are not. Justify your answers. - Name a couple of potential applications (Anwendungen) of PCR. 22 8.7 Funktion verschiedener RNA-Sorten Der molekulare Aufbau der RNA wurde bereits früher besprochen. Die verschiedenen RNA-Sorten einer Zelle sind an der Umsetzung der genetischen Information der DNA beteiligt. Sie sorgen also dafür, dass die in der DNA enthaltene Information letztlich in Proteine (Enzyme), welche den Stoffwechsel steuern, umgesetzt wird: mRNA: messenger-RNA. Bringt die in der DNA enthaltene Information zu den Protein- syntheseorten (………………........………….) im Zytoplasma. Da die DNA den Kern nicht verlassen darf, wird von jedem Gen, dessen Information in ein Protein um- gesetzt werden soll, eine Abschrift hergestellt, eben die mRNA. Diese dient als Vorlage für die Proteinsynthese. mRNA ist also nur so lang wie ein Gen, ist 1- strängig und wird nach relativ kurzer Zeit wieder abgebaut. tRNA: transfer-RNA. Sie bringt Aminosäuren zum Ribosom und sorgt dafür, dass sie, gemäss der mRNA-Vorlage, in richtiger Reihenfolge an die Aminosäurekette ge- hängt werden. Die tRNA hat eine Länge von 75 bis 90 Nukleotiden und ist auf charakteristische Weise 3-dimensional gefaltet wie ein Kleeblatt. Sie enthält viele chemisch leicht abgewandelte Basen (Buch S. 134). rRNA: ribosomale RNA. Sie ist, zusammen mit Proteinen, das Baumaterial der Ribosomen. snRNA: small nuclear RNA. Ist Bestandteil der Spleissosomen (Kap. 8.7). siRNA: small interfering RNA. si-RNAs können mit bestimmten Abschnitten der mRNA Basenpaarungen eingehen. Dadurch wird die mRNA blockiert und schliesslich a b- gebaut. Dieser Mechanismus heisst RNA-Interferenz. siRNAs können also Gene verstummen lassen. Alle RNA-Sorten werden durch Transkription im Kern hergestellt. 23 3-dimensionaler Bau der tRNA: 24 8.8 Transcription in prokaryotes Book p. 132. 1. Warum muss die Information der DNA ins Zytoplasma gelangen? 2. Wie geschieht dies? 3. Was ist ein Gen? 4. Wie lange ist eine mRNA? 5. Wie heissen Start- und Stoppsequenz für die RNA-Polymerase? 6. Wie heissen nicht-codierende Bereiche in eukaryotischer DNA? 7. Wie sehen die Enden der mRNA bei Eukaryoten aus? 8. Beschreiben Sie den Vorgang der Transkription in eigenen Worten. 9. Erstellen Sie eine Tabelle in welcher Sie Replikation und Transkription einander gegenüber stellen (vergleichen). (Skript S. 26 Kap. 8.8.1) The DNA section coding for one gene is "copied into" an mRNA segment by transcription („Überschreiben“). Only one of the two DNA strands contains the correct information: codogenic or template strand (codogener Strang) = matrix strand (Matrizenstrang) = antisense strand. The strands are separated by the same enzyme that synthesises mRNA immediately afterwards: RNA polymerase. There are special recognition features (Erkennungs- merkmale) before and after each gene: in front of the gene is the promoter; this is the site where RNA polymerase binds and starts transcription. At the end of each gene is a terminator. RNA polymerase now finds the promoter with the aid of a transcription factor (Transkriptions- faktor) that binds the polymerase to the correct site: initiation. As the RNA polymerase can bind new nucleotides only to a free 3’ end, the direction of reading is clearly defined. Polymerase now unwinds the two strands continuously, migrates along the cod ogenic strand to the terminator and synthesises a piece of mRNA that is complementary to the DNA in the process: elongation. When synthesis is complete, the mRNA detaches from the polymerase and migrates into the cytoplasm: termination. In prokaryotes, the genes align seamlessly (nahtlos) on the chromosome. If several enzymes are required for a metabolic pathway, the corresponding genes usually follow immediately one after the other. Eukaryotes exhibit a different organisation of their genome. In the first place, long, non- coding sections lie between the genes and, secondly, non-coding regions, introns, are found within the genes. These are cut out by so-called "splicing" („Spleissen“, part of the RNA 25 processing which also includes capping and polyadenylation). The coding regions of the gene, which are part of the final mRNA, are called exons. It is estimated that less than 5 % of human DNA codes for proteins. Transcription in eukaryotes is discussed in the 3 rd year of the course (book p. 136/137). 8.8.1 Replikation vs. Transkription Vorgang Replikation Transkription Ziel Endprodukt Polymerase Startpunkt Eigenschaften und Zielort der Syntheseprodukte Nukleaseaktivität Aufbaurichtung des neuen Stranges Richtung des abzu- lesenden Teil- stranges der DNA Abschluss der Synthese 26 8.9 Genetischer Code In der Basenabfolge der mRNA ist nun die Bauanleitung für ein Protein, d. h. die Reihenfolge der Aminosäuren, enthalten. Da die „Nukleinsäuresprache“ aus 4 Buchstaben besteht, die „Proteinsprache“ jedoch aus 20, wird eine Aminosäure durch drei aufeinanderfolgende Nukleotide bestimmt: Ein Triplett oder Codon. So heisst z.B. das Codewort für Histidin „CAC“ oder „CAU“. „GAA“ bedeutet Glutaminsäure, „AUC“ Isoleucin, etc. Da aus 4 Basen 64 Codons gebildet werden können (4 3), gibt es für die meisten der 20 Aminosäuren mehr als 1 Codon. Man bezeichnet daher den genetischen Code als degeneriert (degenerate). Das Codon AUG bedeutet "Start" (Startsignal für die Proteinsynthese), die Codons UGA, UAG und UAA heissen "Ende" (Abbruch der Synthese). Der genetische Code ist universell (universal), d. h. er gilt, soweit man bis heute weiss, in allen Lebewesen. Nur in Mitochondrien gibt es einige wenige Abweichungen (deviations). 27 **Übung 1: 5‘- A T G A T C G C C -3‘ 3‘- T A C T A G C G G -5‘ → → mRNA → Aminosäurekette 8.9.1 Aufgaben zum genetischen Code 1. In welches Peptid wird folgende Sequenz übersetzt? 5’- U U A G A U G A G C G A C G A A C C C C U A A A A U U U A C C U A G U A G U A G C C A U -3‘ 2. In welches Peptid wird folgender Abschnitt übersetzt? 3‘- C T G G C T A C T G A C C C G C T T C T T C T A T C -5‘ 8.10 Translation Book p. 134/135. The following factors are required for the "translation work" (“Übersetzungsarbeit”): - A template (Vorlage) in nucleic acid language: mRNA - "Interpreter" (“Übersetzer”): tRNA - A "dictionary": genetic code - Place where the process can proceed in a coordinated fashion: ribosomes tRNA has a very specific spatial structure (chapter. 8.7) and possesses two crucial regions for translation: at one end it can bind a specific amino acid, while at the opposite end is a base triplet that is exactly complementary to the codon for the amino acid concerned. This is known logically as an anticodon. The anticodon of tRNA fits like a "plug" ("Stecker") into the 28 "socket" ("Steckdose", => Codon) on the mRNA. Exactly the right amino acid is appended (angehängt) to the "plug" that fits the "socket". Corresponding to the 64 codons, minus the 3 "stop" codons, there are 61 types of tRNA. Protein synthesis itself occurs at the ribosomes. At the 5’ end of the mRNA (with a „cap“), a large and a small ribosomal subunit combine to form a ribosome. The ribosome covers precisely three codons (three triplets) and contains three indentations (Einbuchtungen) into which three loaded tRNA molecules fit (A-, P- and E-site). Amino acid 1 (always methionine, in the A-site => aminoacyl) binds to amino acid 2, which is still connected to its tRNA (in the P-site => peptidyl). Met-tRNA detaches (after being moved to the E-site => exit) and is reloaded with Met in the cytoplasm. The ribosome slides one triplet further and the growing amino acid chain is always appended to the "front" (in the direction of the 3’ end) amino acid. The chain continues to grow until a stop codon appears. There is no tRNA for the stop codon, as a result of which the chain is broken. Several ribosomes usually migrate one after the other along the mRNA, with each ribosome synthesising one protein molecule. Once sufficient protein is manufactured, the mRNA is broken down again. A vital step in translation is the binding of tRNA with exactly the right amino acid. If an error occurs here, the wrong amino acid is incorporated (eingebaut) since there is no longer any possibility for correction in the ribosome. These enzymes that catalyse the "loading" of tRNAs are known as aminoacyl tRNA synthetases. They possess a recognition site for the anticodon and a binding site for the associated amino acid. Translation is an energy-intensive process: ATP is needed to bind the amino acid to the tRNA and GTP is needed for migration of the ribosome. 8.10.1 Hemmstoffe der Transkription & Translation Substanzklasse Substanz Wirkungsmechanismus Antibiotika bindet an prokaryontische 50S- Chloramphenicol Ribosomenuntereinheiten; Breitbandantibiotikum Hemmung der bakteriellen Erythromycin Proteinsynthese durch Bindung an die 50S-Untereinheit der Ribosomen Antibiotikum Hemmung der Translokase → Fusidinsäure Elongationsabbruch Antibiotikum (v.a. gegen gram+) Hemmung der DNA-abhängigen RNA- Polymerase Rifampicin Antibiotikum gegen Tuberkulose & Lepra (Mycobacterien) 29  Bindet an 30S-Untereinheit bei Prokaryonten → Streptomycin Konformationsänderung → Ablesefehlern Antibiotikum, Pestizid bindet an prokaryontische 50S- Tetracyclin Ribosomenuntereinheiten Breitbandantibiotikum Strukturanaloga verursacht Peptidkettenabbruch bei 70S- Puromycin und 80S- Ribosomen nur experimentelle Anwendung Zytostatika Gift des grünen Knollenblätterpilzes (Amanita phalloides) Hemmstoff der Transkription in eukaryonten Zellen Bereits geringe Konzentrationen führen durch Anlagerung an die RNA- Polymerase II B zur Hemmung der prä- α-Amanitin mRNA-Synthese. In weitaus höherer Dosierung wird auch die RNA-Polymerase III blockiert → Hemmung der tRNA-Synthese Auf die Transkription bei den Prokaryonten hat α-Amanitin keine Auswirkungen Ausserdem gibt es noch Stoffe die auf die Synthese von Nukleotiden wirken und solche, welche gezielt gegen Viren eingesetzt werden können (siehe Kapitel Mikrobiologie). 8.11 Mutations Mutations are changes in the genome, chromosomes or the DNA molecule. Not every mutation has consequences for the organism concerned. If the change does not affect the active centre (aktive Zentrum) of an enzyme or does not interfere with the three- dimensional structure of the protein, no consequences are to be expected. In addition, in eukaryotes usually only one of the two alleles is affected. If the mutation is recessive, it is not noticed. Naturally mutations at non-coding sites of the DNA have no effect either. - If the total number of chromosomes of a cell is altered, this is referred to as a genome mutation, for example trisomies and monosomies. See chapter „Genetics“. - A change in chromosome structure is known as a chromosome mutation. Such changes are detectable under the light microscope See chapter „Genetics“. 30 - Changes to the DNA are not microscopically detectable. Such DNA damage and its causes are summarised in a table on p. 139 of the Biology book. DNA damage occurs very frequently. For example, it is known that the smoke from a single inhaled cigarette damages the DNA of lung cells at several thousand sites. The same happens to skin cells from UV light. That this damage usually remains without any effect for a long period of time is due to the fact that it is constantly corrected by repair enzymes in the cell. The repair enzyme "at the base" is DNA polymerase itself: it subjects every newly incorporated nucleotide to a check, constantly excises errors independently and inserts correct nucleotides. However, finished DNA also is constantly checked. To date, more than 50 different DNA repair enzymes have been identified in yeasts; in humans, several more are to be expected. Examples: - Dimers and cross-links (Quervernetzungen) are liberally excised and correctly supplemented (excision-repair). Book p. 140. - Base analogues are identified and replaced. - Single strand breaks (Einzelstrangbrüche) are repaired by ligase. Generally it may be said that damage in only one strand can be remedied without problem since the correct counter-strand can serve as a template. However, damage to both strands can have serious consequences: for example, a double-strand break can result in the loss of the piece of DNA (loss of end piece of a chromosome). Deletion or insertion of a base pair results in frameshifts on reading, which in most cases results in completely non-functional proteins. 8.12 Der Genbegriff Bei Prokaryoten ist der Begriff „Gen“ klar und einfach: Ein Gen ist der Abschnitt auf der DNA, der für ein Protein codiert. Bei Eukaryoten hingegen ist die DNA-Sequenz, die für ein Protein codiert, wegen der Introns nicht durchgehend. Je nachdem, welche Segmente beim Spleissen als Exons über- nommen werden, kann ein Gen für mehrere Proteine codieren: Alternatives Spleissen. Dies dürfte eine Erklärung dafür sein, warum der Mensch mit relativ wenig Genen, schätzungsweise gegen 30'000, ausgestattet ist. Auch eine Mischung von Exons aus voll- ständig verschiedenen Genen (exon shuffling) ist möglich. So gibt es z. B. mehrere 31 Millionen unterschiedliche Antikörper, für die nur etwa 1’000 Gene nötig sind (etwas mehr dazu im Kapitel «Immunologie»). Für Details s. 3. Lehrjahr. Aus den erwähnten Gründen ist das Ein–Gen–ein–Protein–Konzept bei Eukaryoten nicht sinnvoll. Es hat sich folgende Definition durchgesetzt: Ein Gen ist ein Abschnitt (segment) der DNA, der zur Herstellung eines RNA-Moleküls benötigt wird (DNA → RNA → Protein). 8.13 Control of gene activity in prokaryotes A bacterial cell does not offer space for all the proteins that it can translate at the same time. In addition, compartmentalisation is absent in bacterial cells: the only compartment is the cytoplasm. If all possible enzymes were produced constantly, this would result in hopeless confusion. Bacteria must therefore very efficiently express (exprimieren) only those genes that they require at the time. If, for example, they are placed in a medium that contains lactose instead of glucose, the genes for lactose breakdown are induced within a short period of time (synthesis of the corresponding enzymes is turned on) and those for glucose breakdown are repressed (the corresponding DNA segments are blocked). Anabolic metabolic reactions (anabolische = aufbauende Stoffwechselreaktionen) are also regulated: if, for example, the amino acid tryptophan is produced, genes that code for enzymes involved in trp synthesis are turned on. If enough trp is produced, the genes are blocked again. Gene regulation for lactose breakdown and trp synthesis is described on p. 142/143 of the book. Study the theory and answer the following questions: 1. What is an operon? 2. Why does it take a few minutes for E. coli to switch from glucose to lactose nutrition? 32 3. What does "substrate induction" (Substratinduktion) mean? In which metabolic reactions does substrate induction primarily occur? 4. How many different mRNA molecules are produced for the lactose-degrading enzymes? How many different enzymes result from this? How is that possible? 5. What are constitutive genes (konstitutive Gene), what are regulated genes (regulierte Gene)? 6. To which sites on the lac operon do RNA polymerase and the repressor bind? Could the binding sites also be interchanged? 7. How is the repressor of the lac operon activated and deactivated? 8. How is the repressor of the trp operon activated and inactivated? Why does activation/inactivation occur in exactly the opposite way as with the lac operon? 9. Describe in your own words what end-product repression (Endproduktrepression) means and what advantage this process offers for E. coli. 33 8.14 Gentechnik / Genetic engineering Unter Gentechnik versteht man die Übertragung von DNA von einer Art auf eine andere und das Verändern des Genoms (Gene an / aus schalten, Fehler korrigieren etc.). Es werden dadurch Gene exprimiert, die ursprünglich nicht zu dieser Art gehört haben. Die Ziele gentechnischer Methoden reichen von Pflanzen mit erhöhter Widerstandskraft oder höherem Ertrag über Tiere mit veränderten Eigenschaften bis hin zur Heilung menschlicher Erb- krankheiten. Diesen Erwartungen stehen zum Teil erhebliche ethische Bedenken gegenüber. Ethisch unbedenklich sind Bakterien-, Hefe- oder Zellkulturen, die durch den Einbau fremder Gene die entsprechenden Proteine herstellen und sie ins Medium abgeben. Konsultieren Sie das Biologiebuch S. 176-179 & S. 200, sowie die Broschüre „Gentechnik“ und die zur Verfügung gestellten Zusatztexte zur Beantwortung folgender Fragen: 1. Wie werden Fremdgene (foreign genes) in Bakterien eingebaut? 2. Wie werden Fremdgene in pflanzliche und tierische Zellen eingebaut? 3. Stelle Sie eine Liste von gentechnisch veränderten Bakterien, Pflanzen und Tieren zusammen und fügen Sie gegebenenfalls ethische oder ökologische Einwände an. 4. Gentherapie beim Menschen: Was ist bereits Realität, welche Ziele werden verfolgt? 34 8.15 Lernziele „Molekularbiologie“ Thema Biologische Ausgangsmaterialien und Organismen BIO1 07 Molekularbiologie Für eine genetische Untersuchung sollen Primer bestellt werden. Die Sequenz ihres Proteins ist vorgegeben und die Annealing-Temperatur soll 58°C betragen. Welche Sequenz müssen Sie für einen Vorwärts- und einen Rückwärtsprimer bestellen? BIO1 Die Versuche von Griffith, Avery, sowie von Hershey & Chase erklären und deren Bedeutung 07.01 für die Molekularbiologie darlegen können, sowie über die Entdeckung von Watson & Crick Bescheid wissen (experiments of Griffith, Avery, Hershey & Chase, Watson & Crick). BIO1 Die Begriffe Base, Nukleosid und Nukleotid erläutern können (terms base, nucleoside and 07.02 nucleotide). BIO1 Die Basen A, T, G, C und U kennen (bases). 07.03 BIO1 Den Aufbau der DNA beschreiben und das Prinzip der Basenpaarung verstehen können 07.04 (DNA structure and base pairing). BIO1 Den Aufbau der Chromosomen erläutern können (chromosomes). 07.05 BIO1 Den Bau der RNA beschreiben und von DNA unterscheiden können (RNA vs. DNA) 07.06 BIO1 Die unterschiedlichen RNA-Sorten und deren Funktion aufzeigen können (RNAs). 07.07 BIO1 Die Replikation der DNA wiedergeben können (replication). 07.08 BIO1 Die PCR als Replikation in vitro verstehen und mit der Replikation in vivo vergleichen können 07.09 (PCR). BIO1 Den Ablauf der Transkription bei Prokaryoten beschreiben und mit der Replikation 07.10 vergleichen können (transcription). BIO1 Einige gebräuchliche Hemmstoffe der Replikation & Transkription (Antibiotika, Zytostatika) 07.11 nennen und deren Wirkungsweise erklären können (inhibition). BIO1 Den genetischen Code erklären und anwenden können (genetic code). 07.12 BIO1 Den Ablauf der Translation beschreiben können (translation). 07.13 BIO1 Die Ursachen von Gen-Mutationen kennen und die Folgen abschätzen können (gene 07.14 mutations). BIO1 Den Mechanismus des excision repair beschreiben können (excision repair). 07.15 BIO1 Die heute gültige Definition eines Gens erklären können. (definition of a gene) 07.16 BIO1 Genregulation bei Prokaryoten erklären können. (gene regulation in prokaryotes) 07.17 BIO1 Den Einbau von Genen in Prokaryoten wiedergeben können (transformation). 07.18 BIO1 Die Einsatzmöglichkeiten der Gentechnologie in verschiedenen Gebieten beschreiben 07.19 können, insbesondere aktuelle Methoden (genetic engeneering). BIO1 Nutzen und Gefahren der Gentechnologie diskutieren können (pros and cons). 07.20 Buchseiten: 106, 120-124, 126, 132, 134/135, 139/140, 142/143, 175-179, 200 35