1 2 3 4 5 About me: Hey ! I’m Angelica. - Graduated in 2019 with a 97.25 ATAR - 49 Biology study score, 40 PE, 47 English - Completed biomedical science at Monash - Now studying medicine at Monash - Tutor Biology, PE and English at TuteSmart - Love: my cats, long-distance running, paying for overpriced avocado toast, reading in cafés and people watching. 6 What are we covering today Topics to be covered • • • • • Announcements Gene Expression & Regulation Cellular Respiration Photosynthesis DNA Manipulation Questions & Study Tips & Advice Overview Gene Expression & regulation Cellular Respiration Feel free to email me at: angelica@tutesmart.com ☺ Photosynthesis DNA Manipulation Questions Summary 7 Gene Expression What is Gene Expression? • Gene: a particular sequence of DNA bases that code for a specific polypeptide chain • Inherited from parent to offspring • locus = fixed position on a chromosome where a specific gene is located • Gene expression: the transcription and translation of a gene • the process in which the genetic material (DNA) is converted into a functional 3D protein Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 8 Gene Expression Overview Gene Expression & regulation Gene Expression Overall Process Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 9 Gene Expression Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 10 Gene Expression Part 1: Transcription • The process of producing mRNA from a DNA template • Occurs in the nucleus 1. RNA polymerase (an enzyme) binds to the promoter region of the gene to be transcribed on the template strand of DNA 1. The RNA polymerase molecule unwinds the DNA and moves along the template strand ‘reading’ it in a 3’ to 5’ direction whilst synthesising RNA by joining ribonucleotides in the 5’ to 3’ direction (remember strands are anti-parallel) 1. When RNA polymerase reaches the end of the gene (termination sequence), the pre-mRNA molecule will be released 1. The pre-mRNA strand is complementary to the template strand and has the same sequence as the coding strand (except that T is replaced with U) Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 11 Gene Expression Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 12 Gene Expression Part 2: RNA Processing • Eukaryotic cells undergo three important types of posttranscriptional modifications within the nucleus 1. Introns are removed and exons are spliced (joined) together ― ― ― Introns are non-coding regions Exons are coding regions (remember exons are expressed) This means that ‘mature’ mRNA is shorter than pre-mRNA 2. A methyl guanosine cap is added to the 5’ end of the RNA molecule 1. A poly-A tail is added to the 3’ end of the RNA molecule • Once these modifications have taken place, the RNA molecule is mature mRNA and will leave the nucleus and move to a ribosome Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 13 Gene Expression Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 14 Gene Expression • Removing introns from the molecule is important as these regions do not ‘code’ for protein molecules and they will be useless in translation (note that these regions are often important for other reasons) • i.e. One gene may be able to code for more than one protein (or variation of a protein) depending on • which exons are kept in or left out (alternative exon splicing) This may help explain why the amount of proteins the body makes are so diverse despite the amount of DNA present • The 5’ methyl cap is important because it helps initiate the process of translation (recognition at the ribosome) • Both the 5’ cap and the 3’ poly-A-tail protect the RNA strand from damage (e.g. from enzymes) and add stability • The 3’ poly-A-tail assists in export from the nucleus Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 15 Gene Expression Part 3: Translation • Protein synthesis; the process by which a polypeptide molecule is produced from mRNA at a ribosome • How? 1. 1. 1. 1. 1. 1. Overview Once it leaves the nucleus, the mRNA strand migrates to a ribosome The mRNA strand will enter the ribosome at the 5’ end The start codon AUG instructs for translation to begin, directing for the amino acid methionine to start the polypeptide chain Each successive codon in the mRNA will pair up with the anticodon of a tRNA molecule carrying a specific amino acid within the ribosome The process continues with more codons and anticodons pairing, resulting in the amino acids being carried by the tRNA molecules being added to the growing polypeptide chain via peptide bonding (condensation polymerisation) Once a stop codon (UAA, UAG, or UGA which don’t code for an amino acid) is reached, translation will cease and the polypeptide chain will be released Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 16 Gene Expression Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 17 Gene Expression Gene Structure • introns – non-coding sections of a gene • exons – sections of the gene that form mRNA + code for proteins • promoter region – section of gene that RNA polymerase binds to • operator regions are found in prokaryotic operons (we’ll look at these in a few slides!), but are not in eukaryotic genes Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 18 Gene Expression • How come different cells produce different proteins even though every cell contains the same genome? • Cells can essentially switch genes ‘on’ and ‘off’! • By doing so they up- and downregulate the production of certain proteins based on the cell’s specific needs Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 19 Gene Expression How are Genes Turned On + Off? • Transcription factors: DNA binding proteins that bind to specific sequences of DNA (ie. the promoter region) and regulate gene expression by either increasing or decreasing transcription of a gene • Transcription factors called repressors can prevent transcription by binding to the promoter region or operator region of DNA, thereby preventing RNA polymerase from binding and transcribing the DNA Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 20 Gene Expression How are Genes Turned On + Off? • Regulatory genes: genes that code for the production of proteins (such as transcription factors) that regulate gene expression • Structural genes: genes that produce a protein or product that forms part of the structure or the functioning of an organism (essentially every gene except regulatory genes) Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 21 Gene Expression Gene Expression • Signalling molecules (e.g. hormones) that bind to receptors on/within a cell can influence gene expression in that cell • • • • Can activate transcription factors that assist in RNA polymerase binding Can inactivate transcription factors that prevent RNA polymerase binding Can inhibit or ‘switch off’ a particular gene Hormones can also bind to DNA directly to alter gene expression • Gene expression is influenced by cell type, stage of development and the external + internal environment of the cell Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 22 Gene Expression The Importance of Gene Regulation • Different cells can produce different protein products despite containing the same DNA • Genes are only expressed when the product is needed (saves energy and resources) • Producing excess proteins, or proteins when they are not needed, could harm the organism • Some genes are only required to be expressed at certain points in the lifespan (e.g. in embryonic development) • Some genes need to be expressed in all cells at all times • Often referred to as housekeeping genes Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 23 Gene Expression The trp Operon • Operon: a segment of DNA containing a group of genes that are transcribed together (DOESN’T EXIST IN EUKARYOTES) • Separate from the promoter (where RNA polymerase binds) • The trp operon is a prokaryotic example of gene expression regulation • There is a gene in some bacteria, including E. coli, that codes for the production of the protein tryptophan (trp) Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 24 Gene Expression The trp Operon • When too much tryptophan is present, the tryptophan binds to a repressor (type of transcription factor), causing it to bind to the operator region of the gene – this stops transcription from occurring by preventing RNA polymerase from binding to the promoter region • When there is little/no tryptophan, this means that tryptophan doesn’t bind to the repressor, causing the repressor to change in conformation. The repressor then detaches from the operator region allowing transcription to occur (as RNA polymerase can then bind to the promoter of the operon) ― The repressor is coded for by a regulatory gene (trpR) that sits upstream of the tryptophanproducing gene on the operon Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 25 Gene Expression Overview Gene Expression & regulation The trp Operon Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 26 Gene Expression trp Operon - Attenuation • A second method of gene regulation in the • • • • trp operon is attenuation While repressors prevent transcription from starting, attenuation prevents transcription from completing A region called the leader codes for an attenuator sequence • The attenuator sequence forms hairpin structures Within the leader are two tryptophan codons This means to translate the leader we need tryptophan! Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 27 Gene Expression • • Low tryptophan levels: • • The ribosome moves through the leader slowly as we need tryptophan for translation A hairpin structure is created that does not stop transcription – i.e. transcription occurs !!! High tryptophan levels: • • • • trp Operon - Attenuation The ribosome moves through the leader quickly as we have lots of tryptophan to translate the sequence A hairpin structure is created that does stop transcription – i.e. transcription doesn’t occur The ribosome falls off the mRNA and RNA polymerase detaches from the operon Attenuation is possible because transcription and translation occur at the same place (i.e. the cytosol) – think about why this isn’t possible in eukaryotes? • As RNA polymerase moves through the operon, the ribosome can begin translating the mRNA, even though the full strand isn’t completed yet Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 28 Energy transformations Cellular Respiration Photosynthetic Autotrophs Chemosynthetic Autotrophs Synthesise their own organic carbon compounds from inorganic materials, using light as the energy source to do so Synthesise their own organic carbon compounds from inorganic materials, using energy derived from chemical processes Heterotrophs Are reliant on intake and digestion of organic molecules from an external source ATAR Notes Biology Units 1/2 Course Notes Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 29 Cellular Respiration What is Cellular Respiration? • While photosynthesis is the process by which complex organic compounds are made from simple inorganic compounds, how do we use them for energy? • Cellular respiration is how we break down these molecules and turn them into a usable form of energy! Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 30 Cellular Respiration What is Cellular Respiration? • Cellular respiration is a metabolic process whereby ATP is formed in cells from ADP + Pi using glucose as a ‘fuel’ • ATP is a molecule that contains useable chemical energy (in the form of a high energy bond) that can then drive essential reactions within cells ― Without ATP we would die Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 31 CELLULAR RESPIRATION Cellular Respiration “What’s the most important thing you remember from school?” *inhales* Overview Gene Expression 💔 THE MITOCHONDRIA IS THE OF THEDNA CELL CellularPOWER Respiration HOUSE Photosynthesis Manipulation Questions Summary 32 Cellular Respiration • • Overview Pathways Aerobic respiration: ― Requires oxygen ― Produces lots of ATP ― Occurs in the mitochondria ― Occurs slower Anaerobic respiration: ― Doesn’t use oxygen ― Produces little ATP ― Occurs in the cytosol ― Occurs faster Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 33 Cellular Respiration Anaerobic Respiration (Fermentation) • The metabolic pathway that is followed in the absence of oxygen (or the absence of mitochondria e.g. in RBCs) • Occurs in the cytosol Animals 2X LACTATE (3C) + NAD+ 2x PYRUVATE (3C) + NADH Glucos e (6C) Yeasts 2X ETHANOL + 2X CO2 + NAD+ 2 ATP • Essentially this stage is just glycolysis, then the cell processing the products Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 34 Cellular Respiration Aerobic Respiration 2 x ATP NADH H2O • Note the coenzymes (electron carriers) NADH and FADH2, these are very important later on! Overview Gene Expression O2 2 H AD N + H D A F 32–34 x ATP 2 x ATP 2CO2 Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 35 Cellular Respiration • The first stage of both the aerobic and anaerobic pathway of respiration • Involves 1 molecule of glucose (6C) being broken down into 2 molecules of pyruvate (3C) • Occurs in the cytosol of the cell • This stage does not require oxygen (it is anaerobic) • Produces net 2 ATP Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 36 Cellular Respiration Overview Gene Expression Cellular Respiration Inputs + Outputs Photosynthesis DNA Manipulation Questions Summary 37 Cellular Respiration Mitochondria • Site of the Krebs cycle and electron transport chain (aerobic respiration) • 2 membranes (inner membrane is highly folded & convoluted) • Contains mitochondrial DNA • Cells that need lots of ATP (like muscle cells) have lots of mitochondria • Red blood cells have no mitochondria ― What does this imply? Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 38 Cellular Respiration • Step 1: Each pyruvate (3C) is converted to acetyl CoA (2C) and 1 CO2, the loss of electrons reduces NAD+ to NADH (Known as the pre-step/intermediate step) CoA Pyruvate NAD+ Acetyl CoA NADH CO2 • Step 2: The acetyl CoA enters the Krebs cycle in the mitochondrial matrix and with each ‘turn’ of the cycle 1 Acetyl CoA gives rise to 2 CO2 molecules, 1 ATP, 3 NADH and 1 FADH2 (TWO TURNS PER GLUCOSE) Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 39 Cellular Respiration It gets complicated But all we need to know is… Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 40 Cellular Respiration Inputs + Outputs • 2 pyruvates, therefore 2 acetyl CoA’s and so the cycle happens twice • Inputs (per glucose) are: • 2 x pyruvate (specifically 2 x Acetyl CoA)• With each cycle we get: (this happens twice per glucose!) • NAD+ • 2 ATP • FAD • 6 NADH • 2 ADP + Pi • 2 FADH2 • 4 CO2 Occurring in the matrix of the mitochondria Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 41 Cellular Respiration • Protein complexes sit in the cristae of the mitochondria which have the ability to receive and donate electrons via carrier molecules (NADH & FADH2) • Electrons are passed between these complexes like a chain • The movement of electrons does work (using spin energy), enabling the protein complexes to actively pump H+ into the intermembrane space creating a proton gradient (potential energy) • H+ ions therefore move down their concentration gradient into the ATP synthase enzyme, enabling the enzyme to synthesise ATP from ADP + Pi (producing 32-34 ATP molecules) • Oxygen acts as the final electron acceptor and is converted to H2O Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 42 Cellular Respiration Electron Transport Chain CRISTAE (folded inner membrane of mitochondrion) e- e- e- e- e - Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 43 Cellular Respiration ETC It’s easy to get confused with how much we need to know about this but essentially… Electron Carrier molecules give up electrons and hydrogen at the cristae of the mitochondria The electrons are accepted by and passed along a series of electron acceptors, on the cristae The interaction between the electrons and protein complexes facilitates the production of ATP Oxygen then captures the electrons which then combines with hydrogen to form water Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 44 Cellular Respiration Inputs + Outputs • Inputs (per glucose): ― ― ― ― NADH FADH2 ADP + Pi 6 O2 • Outputs (per glucose): ― ― ― ― NAD+ FAD 32 – 34 ATP 6 H2 O • Site: Cristae of the mitochondria Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 45 Cellular Respiration Overall Reaction OVERALL: Word eqn. Glucose + oxygen Chemical eqn. 6O2 + C6H12O6 carbon dioxide + water 6CO2 + 6H2O Total ATP Yield 36-38 ATP (memorise) Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 46 Cellular Respiration Overview Gene Expression Cellular Respiration Summary Photosynthesis DNA Manipulation Questions Summary 47 Cellular Respiration • The process in which glucose is broken down to form ATP There are two types of cellular respiration Aerobic respiration Overview Gene Expression Cellular Respiration Anaerobic respiration Photosynthesis DNA Manipulation Questions Summary 48 Cellular Respiration cle al y i s C ndr b o e Kr och trix t a Mi m Gl yc o cy In lys is to pl as m Electron Transport Chain Cristae (mitochondrial membrane) Overview Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 49 Cellular Respiration Overview Gene Expression Anaerobic Respiration Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 50 Cellular Respiration Overview AEROBIC ANAEROBIC Oxygen required No oxygen required Mitochondria Cytosol Water + CO2 A: Lactic acid P/Y: Ethanol + CO2 More ATP Less ATP Gene Expression Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 51 ATP Photosynthesis Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 52 Photosynthesis Photosynthesis 🌱 Overview Gene Expression & regulation 🌱 Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 53 Photosynthesis Photosynthesis • The process in which light energy is transformed into chemical energy Stroma: Light Independent Stage Thylakoid: Light Dependent Stage Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 54 Chloroplast Photosynthesis • The organelle that is the site of photosynthesis • An outer membrane and an inner membrane • The grana: stacks of thylakoid disks • site of the light dependent stage • The stroma: fluid matrix • site of the light independent stage Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 55 Chloroplast Photosynthesis • Thylakoids within their membranes contain photosynthetic pigments known as chlorophyll • These are the photosynthetic pigments which absorb the light energy used in photosynthesis • There are various types of chlorophyll such as chlorophyll a and chlorophyll b • These different types of chlorophyll absorb different wavelengths of light Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 56 Rubisco Photosynthesis • Rubisco is a key enzyme involved in the Calvin Cycle • It’s role is to fixate the carbon molecule from CO2 into an organic molecule that the plant uses for energy storage (which eventually becomes glucose) • During the Calvin Cycle, Rubisco fixates carbon to become the organic molecule G3P (a three-carbon molecule) • i.e. half of a glucose molecule – two G3P molecules make one glucose • during this fixation process CO2 reacts with RuBP (five-carbon molecule) to make a six-carbon molecule that splits into two G3Ps! Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 57 Light Independent Reaction Photosynthesis Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 58 Photosynthesis Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 59 Summary Photosynthesis STAGE INPUTS OUTPUTS SITE LIGHT DEPENDENT Water, ADP + Pi, NADP+ Oxygen, NADPH, ATP Grana LIGHT INDEPENDENT Carbon dioxide, ATP, NADPH Glucose, ADP + Pi, NADP+ Stroma Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 60 Photosynthesis Efficiency Photosynthesis • Most plants undergo photosynthesis via the pathway we’ve just looked at • These are C3 plants • Under typical conditions photosynthesis works well, however in C3 plants, it can sometimes be inefficient • This is due to a process called photorespiration, which involves the enzyme Rubisco • Rubisco’s role in the light independent stage is to fixate carbon, however during photorespiration, it instead fixates oxygen – this wastes energy and uses up carbon molecules, making photosynthesis inefficient • Photorespiration occurs when carbon dioxide levels are low Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 61 C4 Plants Photosynthesis • C4 plants (e.g. corn) are usually found in hotter areas • Therefore, to minimise water loss C4 plants often have their stomata closed • Having closed stomata lowers the amount of CO2 entering the plant, leading to photorespiration • To combat this, C4 plants separate the light dependent and independent stages into different cells • In a mesophyll cell, CO2 is fixed into a molecule called malate • Malate is then transported into a bundle-sheath cell, where it releases CO2 that is fixated by Rubisco Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 62 C4 Plants Photosynthesis Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 63 CAM Plants Photosynthesis • CAM plants function in a similar way to C4 plants, and are adapted mainly to dry environments • Instead of separating locations, CAM plants separate the light dependent + independent reactions over time • Stomata are open at night, when it is cooler and more humid and this is when CO2 enters the plant • It is then converted into malate, which is stored in the plant until the daytime • During the day malate releases CO2 which Rubisco fixates during the Calvin Cycle Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 64 CAM Plants Photosynthesis • CAM plants can close their stomata during the day and still have a supply of CO2 for Rubisco – preventing photorespiration from occurring Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 65 What are Endonucleases? DNA Manipulation • Special enzymes generated by bacteria, that cut through DNA at specific nucleotide sequences • Also known as restriction enzymes • These enzymes are produced by bacteria to protect themselves from bacteriophages (viruses that infect bacteria) • Molecular biologists can use these enzymes to manipulate DNA for many different purposes (e.g. genetic cloning) How would endonucleases protect bacteria against viruses? Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 66 Endonucleases in Bacteria DNA Manipulation Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 67 How do Endonucleases Work? DNA Manipulation • Restriction enzymes cut DNA at certain recognition sequences that are specific to that particular enzyme by breaking the covalent bonds between nucleotides • The recognition sites are usually palindromic Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 68 Blunt + Sticky Ends DNA Manipulation Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 69 Different Restriction Enzymes DNA Manipulation You do NOT have to memorise any of these! EcoRI, BamHI and HindIII are commonly used in exam questions. Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 70 Endonucleases DNA Manipulation Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 71 What are Ligases? DNA Manipulation • DNA ligases join together segments of DNA by catalysing the formation of phosphodiester bonds between nucleotides (the bonds which hold the DNA ‘backbone’ together). • You can essentially think of ligase as ‘glue’ for DNA. • Therefore ligase is very important if you want to manipulate DNA! Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 72 What are Polymerases? DNA Manipulation • DNA polymerase is an enzyme that is responsible for forming new DNA strands by joining nucleotides (essential for DNA replication, but also PCR) Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 73 CRISPR-Cas9 DNA Manipulation • A DNA manipulation tool that involves directly editing genes within an organism • CRISPR-Cas9 is an enzyme found in bacteria, that functions somewhat like an immune system • we’ll look at human immunity in Unit 4! • CRISPR-Cas9 works in bacteria by recognising viral DNA and cleaving it (thereby protecting the bacterium from the virus) • Scientists have used this mechanism to edit genomes by making CRISPRCas9 cleave DNA at a specific location Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 74 CRISPR-Cas9 in Bacteria DNA Manipulation • CRISPR-Cas9 acts like an immune system by ‘remembering’ viral DNA that has previously entered the bacteria • When viral DNA enters the cell for the first time, a sequence of the DNA (spacer) is incorporated into the bacterial genome – within the CRISPR array • If this same viral DNA enters the cell at another time, the bacterium will recognise the DNA due to the spacer that matches it • This DNA spacer will be transcribed, becoming the guide RNA • This guide RNA joins the Cas9 enzyme to form the Cas9 complex, which will find the corresponding section of viral DNA and cleave it, effectively destroying the viral DNA and preventing infection Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 75 Applications in Genome Editing DNA Manipulation • • We can also use this system to edit genes! • Scientists create the guide RNA (complementary to the gene of interest) • The Cas9 complex will find the gene in the cell and cleave it • From here, the options are endless – cutting the gene tends to inactivate it, but we can also insert a gene here, delete a section of a gene and more! In bacteria, the guide RNA matches the viral DNA to be cleaved, however we can create a guide RNA with whatever sequence we want – for example a gene responsible for a hereditary disease Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 76 PAM Sequence DNA Manipulation • We discussed endonucleases a few slides ago, and how they know not to cleave DNA belonging to the bacterium • • • • The CRISPR-Cas9 system has a similar safeguard – the PAM sequence This is how Cas9 knows to cleave viral DNA but not the spacer in the CRISPR array Cas9 will only cleave DNA that is followed by a PAM sequence (2-6 nucleotides) • • bacterial DNA is methylated, whereas foreign DNA isn’t, so endonucleases only cut DNA they’re supposed to the DNA spacers within the CRISPR array are not followed by a PAM sequence, so Cas9 doesn’t cleave this DNA! Therefore, when scientists create a guide RNA, they first have to make sure that there is a PAM sequence next to the gene they’re targeting Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 77 Concerns DNA Manipulation • CRISPR-Cas9 is a fairly new discovery and its applications in genome editing are still being studied • Some concerns are: • If a gene is inserted, what happens when it’s done it’s job and is no longer required? • What if Cas9 cleaves at unwanted locations? • Will it work in the long-term? For example, people with chronic diseases will constantly • • need the gene to be working. What if a gene has unknown influence on a certain characteristic/function, and is inactivated? What if the mutations caused by CRISPR-Cas9 lead to cancer? • The main issue is potential off-target effects Overview Gene Expression & regulation Cellular Respiration Photosynthesis DNA Manipulation Questions Summary 78
