Cellular and Molecular Principles of Life Notes PDF

Cellular and Molecular Principles of Life Criteria of life Evolution through natural selection. Separated from their environment by a boundary. Metabolically active and use their metabolism to maintain themselves, grow, and reproduce. Anything made of cells meets the criteria = living. Vi...

Cellular and Molecular Principles of Life Criteria of life Evolution through natural selection. Separated from their environment by a boundary. Metabolically active and use their metabolism to maintain themselves, grow, and reproduce. Anything made of cells meets the criteria = living. Viruses meet the first two, however only meet the third when inside cells = debates over 'living', cycle between non-living and living. Nucleic acids DNA and RNA molecules are polymers consisting of a sugar- phosphate backbone and sequence of bases. DNA is in a double-helix with two anti-parallel stands and joined by hydrogen bonds formed by complimentary base pairing. Information is always read from the 5' end towards the 3' end. Amino acids Common structure with a carboxyl group and amino group. Variation is a result of differences in the R group in the side chain. 20 amino acids encoded in the genome and used to make proteins. Lipids Key function is membrane formation (also involved in energy storage and cell communication). Generally hydrophobic, but can be amphiphilic (e.g. phospholipids with hydrophilic head and hydrophobic tail). Sugars Key function is as a source of energy (also can be used for structural support in cells, usually in the form of complex carbohydrates). Glucose is metabolised to form ATP. Genes - introduction Most of the genome is made up of non-coding DNA, including: o Introns o Regulatory regions o Retroviral sequences (transposons) o Telomeres DNA strands are always read from the 5' end to the 3' end. Endosymbiotic Theory Infoldings in the plasma membrane of an ancestral prokaryote gave rise to endomembrane components, including a nucleus and endoplasmic reticulum. In a first endosymbiotic event, the ancestral eukaryote consumed aerobic bacteria that evolved into mitochondria. In a second endosymbiotic event, the early eukaryote consumed photosynthetic bacteria that evolved into chloroplasts. LECA LECA = Last Eukaryote Common Ancestor. Would have contained: o Nucleus o Mitochondria o Golgi o Lysosomes o Cytoskeleton Evolved from the First Eukaryote Common Ancestor (FECA) which emerged from the endosymbiosis of archaea and proteobacteria. Central dogma Information cannot be transferred from protein to protein, or from protein to nucleic acid, but can be transferred between nucleic acids and from nucleic acid to protein. The translation of RNA into protein is unidirectional. Nucleotides - structure Nucleotides are made up of a: Phosphate group(s) o Monophosphate / diphosphate / triphosphate o Ionized hydroxyl group Five-carbon (pentose) sugar o Ribose (OH) o Deoxyribose (H) Nitrogenous base o Purines (2 rings) ▪ Adenine ▪ Guanine o Pyrimidines (1 ring) ▪ Cytosine ▪ Uracil ▪ Thymine Nucleotides - strands Polynucleotide chains have nitrogenous bases linked to a sugar- phosphate backbone. Nucleotides are linked by phosphodiester bonds. Bases form hydrogen bonds with the antiparallel chain in the formed double helix: o C and G form 3 bonds o A and T form 2 bonds ▪ Complimentary base pairing involves the pairing between a pyrimidine and a purine. DNA - structure The B-form (main form) of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. Double helix has a major (wide) groove and a minor (narrow) groove. The grooves provide access to the bases within the helix without unwinding the DNA - proteins involved in transcription, replication, and repair recognize and bind specific sequences in DNA by "reading" the edges of the base pairs exposed in the grooves. The major groove is more accessible due to its larger size, allowing proteins to interact with a wider area of the DNA base pairs. DNA vs RNA DNA RNA - Replicates - Replicates - Stores genetic information - Stores genetic information - ALSO converts genetic information contained within DNA to a format used to build proteins 2 strands 1 strand Much longer polymer than RNA Variable in length, but much shorter than DNA Sugar = deoxyribose (contains one less hydroxyl Sugar = ribose group) Bases = A,T,C,G Bases = A, U, C, G Chromatin DNA is packed with proteins (histones) in a highly compact shape – chromatin. Nucleosomes = complexes of DNA and 8 histones. Histones are positively charged, DNA is negatively charged. Euchromatin = open conformation, chromatin unwound from histones allowing for DNA transcription. Heterochromatin = closed conformation, chromatin is tightly wound around histones. Chromatin remodelling is affected by factors: o Histones variants. o Histone post-translational modification (e.g., methylation, acetylation). o ATP-dependent chromatin remodeling complexes. Cell cycle Semi-conservative DNA replication DNA replication accomplished by separation of the strands of a parental duplex, each strand then acts as a template for the synthesis of a complementary strand. Bidirectional: replication forks move towards each other, many origins of replication. Linear: o Replication progresses in the 5' to 3' direction. o Initiator proteins separate DNA strands for access of the DNA helicase. o DNA helicase unzips the helix by breaking the hydrogen bonds. o Topoisomerase unwinds the DNA molecule. o Primase synthesises short RNA stretches (primers) which provide a 3'-OH group to start replication. o Elongation requires free dNTPs (deoxynucleoside triphosphate – free nucleotides) and the action of DNA polymerases. dNTPs – as each monomer joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate. Leading strand = 3' to 5' direction, continuous synthesis. Lagging strand = 5' to 3' strand (in 3' to 5' direction), therefore discontinuous synthesis, ligase enzymes join Okazaki fragments. The combined action of exonuclease (enzymes that removes nucleotides – the ones that are laid down by primase to form short RNA primers) and DNA polymerase catalyses the replacement of RNA primers (one on leading strand, multiple on lagging strand) with DNA dNTPs. Replication accuracy and mutations DNA polymerases also possess: o Proofreading activities o Exonuclease activities DNA repair mechanisms: o Base Excision Repair (BER) = single-strand break / single-base damage. o Nucleotide Excision Repair (RER) = bulky lesions / crosslinks. o Mismatch Mediated Repair (MMR) = base mismatch. o Homologous Recombination (HR) = double-strand break. o Non-Homologous End-Joining (NHEJ) = double-strand break. Uncorrected errors = mutations. Telomeres Usual replication machinery provides no way to complete the 5' ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends (not a problem with circular prokaryotic chromosomes). Telomeres = nucleotides sequences at the ends of eukaryotic chromosomal DNA that postpone the erosion of genes near the ends of DNA molecules. Eukaryotic gene structure Regulatory regions: Nucleotide sequences that can increase / decrease gene expression – located far from the regulated gene. o Promoter = sequence of DNA where proteins bind to initiate transcription. Coding regions: o Transcription start site o Exons = code for functional products. o Introns = noncoding sections that are spliced out before the RNA molecule is translated into a protein. o Termination region Gene expression = the process of producing RNA and proteins from a gene. Ribozymes Ribozymes = catalytic RNA – RNA molecules that function as enzymes. "Self-splicing / self-cleaving" - make precise cuts in RNA sequences. Roles include: Cutting / binding sequences. Replicating RNA molecules. Facilitating the creating of bonds in proteins. RNA world hypothesis = chains of RNA may have been the first things to replicate and evolve. Transcription - overview Elements required for transcription initiation: o Starting DNA template o Transcription unit (promoter, RNA coding sequences, terminator) o Machinery for transcription (RNA pol II and up to 50 other proteins) o rNTPs Transcription starts in a 'bubble'. Complementary strand synthesised in 5' to 3' direction, occurs along template strand in 3' to 5' direction. Genes transcribed from the template strand (information on which this is = promoter). Transcription - unit Transcription unit = promoter, RNA coding region, terminator (the fundamental DNA segment responsible for producing RNA and enabling gene expression). Upstream = before the starting point of transcription, closer to the promoter (- nucleotides). Downstream = after the starting point of transcription, closer to the terminator (+ nucleotides). RNA polymerases Type Cellular location Transcription products I Nucleolus 5.8S, 18S, 28S rRNAs II Nucleoplasm mRNA, snRNAs III Nucleoplasm tRNA, 5S rRNA Mitochondrial Mitochondria Mito RNAs Transcription - initiation The transcription machinery recognises and binds to the promoter to start synthesis from DNA (no primer needed). Regulation: o Accessory proteins (general transcription factors) are required for RNA polymerase to recognise the DNA sequence. o Regulatory proteins bind to DNA to modify chromatin structure. o Regulatory promoter also binds transcription factors and regulates the speed of transcription. o TFs can also bind enhances / silencers to regulate transcription (usually located upstream). Abortive initiation: o Phase that occurs during transcription initiation. o Involves the repeated synthesis and release of short RNA transcripts as a result of RNA polymerase being unstable and releasing from the promoter. o It ends when RNA polymerase escapes the promoter and transitions into elongation, synthesizing a full RNA transcript. Transcription – basal apparatus Basal apparatus / general transcription machinery = complex of proteins and enzymes needed for transcription initiation, made up of: o RNA polymerase (synthesises mRNA). o Transcription factors (position RNA polymerase on promotor and involved in transcription initiation). o Mediator complex (facilitates RNA polymerase activity). TATA box: o TFIID is the first general transcription factor to bind to the core promoter. o The TATA-binding protein (TBP) subunit of TFIID recognizes and binds directly to the TATA box. o This interaction causes a local bend in the DNA, helping to recruit other components of the basal transcription apparatus Transcription - elongation The RNA polymerase moves downstream along the template and synthesises mRNA, adding nucleotides at the 3'. DNA moves through a channel in RNA polymerase and makes a sharp turn at the active site. The strands are separated and the newly forming RNA molecule: o Continues growing in the 5' to 3' direction. o Runs through another cleft in the RNA pol II. Changes in the conformations of certain flexible modules within the enzyme control the entry of nucleotides to the active site. Transcription - termination There is no specific termination sequence for RNA pol II – continues synthesising after end of coding sequence. Pre-mRNA is cleaved while RNA pol II is still transcribing. Extra RNA stretch is degraded in the 5' to 3' direction by Rat1 (exonuclease enzyme). Termination occurs when Rat1 reaches the polymerase. MRNA - structure 5' cap: o 5' end. o Guanine nucleotide added. o Methylation of nitrogenous base. o (Sometimes) methylation of the sugar in 1st and 2nd nucleotide. o Cap added immediately after the initiation of transcription (only in RNA pol II transcripts). o Increases mRNA stability, regulates intron removal, important for translation. Poly A tail: o 3' end. o Long chain of adenine nucleotides added via polyadenylation. o Occurs after cleavage. o Contributes to mRNA stability, regulates gene expression, helps attachment to ribosomes for translation. Splicing: o Mediated by the spliceosome. o 5' splice site is cut first, lariat formed. o 3' splice site is cut and two exons are covalently attached. o Intron is released as a lariat and degraded. o Alternative splicing = same DNA molecule can be spliced in different ways to generate different mRNA products (therefore proteins). Nuclear export of mRNA Mature mRNA associates with specific RNA-binding proteins to form a ribonucleoprotein complex (mRNP). The nuclear pore complex (NPC) is a large protein channel embedded in the nuclear envelope, facilitating transport between the nucleus and cytoplasm. The mRNP interacts with the NPC through export receptors and moves through the pore. Regulation: Only properly processed mRNAs are exported - defective or incompletely processed mRNAs are retained in the nucleus and degraded. Certain mRNAs may be exported in response to specific cellular signals or stress conditions. Primary structure Primary structure = sequence of amino acids in a protein. Peptide bonds form between the carboxyl group and amine group of amino acids (condensation reaction). 3D folding Secondary structure: o Hydrogen binding of the peptide backbone causes folding into a repeating pattern. ▪ Alpha helix ▪ Beta pleated sheet Tertiary structure: o 3D folding as a result of side chain interactions. Quaternary structure: o Protein consisting of more than one amino acid chain. Proteins can also contain discrete domains = functional units. Genetic code Codon: triplet RNA code. 64 possible codons: o 3 stop codons (UAA, UAG, UGA) o 61 sense codons (initiation = AUG) Degenerate = more than one codon may specify a particular amino acid. Codons must be read in the correct reading frame (correct groupings) in order for specific polypeptides to be produced. Transfer RNAs - purpose tRNA = transport specific amino acids to complementary codon on ribosome. Each tRNA is only able to bind one amino acid. o Specific nomanclature – tRNAPro is the tRNA bound with proline. Transfer RNAs - structure The secondary structure of tRNA looks like a clover leaf and tertiary structure is like an inverted ‘L’ shape – the folded structure is formed due to hydrogen bonding between complementary bases. Clover leaf structure of tRNA: The secondary folded structure of tRNA has three hairpin loops, which give it an appearance of three- leafed clover. The main constituents of tRNA are: o Acceptor arm - the 3’ terminal ends with a specific sequence which an amino acid attaches to the hydroxyl group of. o D Loop – helps in folding. o Anticodon Loop -contains the complementary codon present on mRNA for the amino acid it carries, the unpaired bases of anticodon loop pair with the mRNA codon (each codon is identified by a specific tRNA). o TΨC Loop - plays a significant role in the overall structure and stability of tRNA. o Variable Loop - present between the TΨC loop and the anticodon loop, helps in the recognition of the tRNA molecule.  Prokaryotic ribosomes Eukaryotic ribosomes Ribosomal RNA Large subunit (LSU) 50S 60S Small subunit (SSU) 30S 40S Assembled ribosome 70S 80S Ribosomes are complexes made up of more than 50 RNA molecules and proteins. Made in the nucleolus. Location of translation from mRNA to proteins. 2 eukaryotic rRNA genes: o One codes for 28S, 18S, and 5.8S, which is processed post transcription – 28S and 5.8S form part of the large subunit, while 18S is separated to form the small subunit. o The other codes for 5S, part of the large subunit which is assembled in the nucleoplasm (transcribed by RNA Pol III, rather than RNA Pol I). Translation - overview Ribosomes bind near the 5' of mRNAs. Synthesis begins at the amino end of the protein (N-terminus), and new amino acids are added at the carboxyl end (C- terminus). Stages: o tRNA charging o Initiation o Elongation o Termination Translation – tRNA charging Binding of tRNAs to amino acids. Shared CCA sequence at 3' of all tRNAs – carboxyl group of amino acid covalently bonds to the nitrogenous base of A. Aminoacyl-tRNA synthetases = enzymes that recognise the complementary tRNA and amino acids: o tRNA and cognate amino acid enter the active site of the specific synthetase. o Using ATP, the synthetase catalyzes the covalent bonding between the amino acid and the tRNA. o The tRNA, charged with the amino acid, is released by the synthetase. Translation – initiation Assembly of the machinery at the ribosome. 1. mRNA binds the small subunit of the ribosome. o SSU and LSU need to be separate for the mRNA to bind to the SSU. 2. Initiator tRNA binds to the mRNA (anticodon-codon binding). o Pre-initiation complex composed of SSU, Met-tRNAiMet, and initiation factors (at least 12) recognise and bind the 5' cap in the mRNA. o Initiation factors: ▪ Prevent binding of LSU to SSU, recognise and bind to 5' cap, recruit and initiate tRNA, bind initiator tRNA and intiator codon, final LSU binding. o Pre-initiation complex scans the mRNA until the start codon is found. o Initiation codon is surrounded by a consensus (Kozak) sequence which helps recognition. o Codon and anticodon (tRNA) bind. o Initiation factors are released. 3. The large ribosomal subunit joins the complex. Translation – pioneer round Cap-binding complex (CBC) promotes a pioneer round of translation to check for errors. CBC is then substituted with e(eukaryotic)IF(initiation factor)-4E for continuation. Translation – elongation Elongation of the polypeptide chain through addition of new nucleotides at the C-terminal. Ribosomes have 3 possible tRNA binding sites: o Aminoacyl (A) - entry. o Peptidyl (P) - where initiator tRNA binds. o Exit (E). Steps in elongation: Charged tRNA (has an attached amino acid) binds to the A site mediated by elongation factor eEF1a (protein that ensures correct binding to the A site). eEF1a is active when bound to GTP (molecule that provides energy). The anticodon of tRNA pairs with the complementary codon on the mRNA strand (tRNA-mRNA pairing). GTP is hydrolysed to GDP, releasing energy and converting eEF1a into its GDP-bound inactive form (which then dissociates from the ribosome). Recycled back to active GTP-bound form by other eEFs. A peptide bond is formed between the amino acids in the A and P sites, catalysed by the 28S rRNA LSU (acts as a ribozyme). After the peptide bond is formed, the tRNA in the P site releases the amino acid (now connected to the amino acid in the A site). Translocation occurs – the process where the ribosome shifts along the mRNA by one codon in the 5' to 3' direction after the polypeptide is formed. eEF2 facilitates translocation as it binds to GTP and uses energy from hydrolysis (GTP -> GDP) to push the ribosome forward. THE RIBOSOME MOVES – NOT THE mRNA AND tRNA. The uncharged tRNA that was previously in the P site is now in the E site which results in it being released into the cytoplasm. The tRNA is recharged by binding a new, corresponding amino acid. The process repeats as a new charged tRNA molecule enters the A site to continue elongation, continuing until the ribosome reaches a stop codon. Translation – termination Protein synthesis ends at the stop codon and the machinery is released. There is no tRNA for the termination codon and therefore the A site is left empty. Release factors mediate the final steps: o ERF1 is required for the recognition of the stop codon. o ERF3 supports the cleavage of the tRNA-polypeptide bond using GTP. o Other RFs mediate the release of tRNA and mRNA and the ribosome dissociation. Roles of the cytoskeleton Provides strength Controls cells shape Transport system Cell division Connect cells in tissues Allow cell movement Actin Structure: Actin molecules are made up of actin filaments (sometimes called microfilaments). Actin filaments are 5-9nm in diameter. Have a plus end and a minus ends – actin is recycled as monomers are cleaved from the minus ends and assembled on the plus ends (process requires ATP). The plus end is usually pointing outwards towards the plasma membrane in order to push the membrane and allow movement. Roles: They provide strength and support to the cell. Highly dynamic – can grow, shrink, branch, form new networks to allow cells to respond to changes in environment. Intermediate filaments Structure: More diverse than actin and microtubules, composed of a wider range of proteins. Size between actin and microtubules. Roles: Provide mechanical strength. Used to connect cells in tissues. Form the nuclear lamina – beneath the membrane that helps to anchor chromosomes and the nuclear pore complex. Microtubules Structure: Hollow tubes Have a plus and minus end, like actin. Minus ends of all microtubules localise to the Microtubule Organising Centre (MTOC), also called a centrosome, located near the nucleus. Pericentriolar material (proteins) 'stick' microtubules onto centrioles. Both grow AND shrink from the plus end. Monomers of the protein tubulin are added to the plus end then capped when fully grown. The cap can be removed in order to shrink the microtubule as tubulin can dissociate from the plus end. Plus ends point towards the plasma membrane. Roles: Operate throughout the cell for transport – remodel to allow the cell to send organelles / vesicles to new parts of the cell. Used during cell division for formation of mitotic spindle. Cell migration Chemotaxis: Cell receives a signal. The cytoskeleton can rapidly change shape as filaments dissassembled then reassembled at new sites in order to respond to the signal (move towards / away from it). Flagella: Multiple microtubules are arranged down the length of flagella / cilia as 'doublets'. Along the doublets are motor proteins called dynein. Doublets can be crosslinked with nexin (protein). Cross-linked doublets prevents motor proteins from walking, instead their energy is used to bend the microtubules. Cilia: Similar to flagella but tend to be shorter and beat in a coordinated manner. Standard light microscopy Staining: Hematoxylin & Eosin stain – commonly used in histology, acidic structures stained purple (e.g., nucleus), basic structures stained pink (e.g., cytoplasm and cell walls). Azan trichrome stain – nuclei stained red, good for staining connective tissue and epithelium as collagen and basement membrane stained blue. Electron microscopy Electron microscopes can't produce colour images (can be artificially coloured – pseudocolours). Can't be used to view live cells due to the need for a vacuum. Fluorescence microscopy Fluorescence = the process by which one wavelength of light (colour) is absorbed and another is emitted. Fluorescence microscopy = type of light microscopy but only uses part of the light spectrum and relies on fluorophores to emit light when stimulated. Fluorophores = molecules attached to antibodies which bind to specific receptors on cells, 'staining' the desired cells. Fluorophores can be used to visualise live cells if staining the surface, however can only enter the cells when dead in order to stain inner structures. Green fluorescent protein (GFP): o Jellyfish Aequorea victoria has a single protein for fluorescence. o Mutants of GFP were created that folded correctly and worked efficiently at 37C. o Fusion proteins can be created as the GFP gene can be inserted before the stop / after the start codon for a gene of interest (separated by a spacer sequence). o Other fluorescent proteins have been isolated and a spectrum of colours has been created. Limitations of using fluorescent proteins: o Photobleaching: proteins can be destroyed by high intensity laser light. o Phototoxicity: toxicity caused by high-intensity laser light. o Overexpression: tagged genes often expressed at high levels for good signal, however excess proteins may be handled by cells differently to normal levels. o Protein folding: fluorescent proteins are quite big and therefore may affect folding and therefore function. o Cellular distribution: large protein size may also impact distribution, especially for membrane proteins. Conversion of light to chemical energy Photosynthesis: Carbon dioxide + water --> glucose + oxygen 6CO2 + 6H2O -(light energy)-> C6H12O6 + 6O2 The great oxygenation event 1. Cyanobacteria produce oxygen. 2. Mass extinction event as oxygen is toxic. 3. Oxygen reacts with methane to form carbon dioxide and water. 4. Reduction in methane resulted in the great glaciation event as methane was the greenhouse gas that was maintaining warmer temperatures. Chloroplasts Double membrane (outer membrane, intermembrane space, and inner membrane). Thylakoid membranes are folds of the inner membrane that stack into grana, location of light-dependent reactions. Stroma is the location of light-independent reactions. Pigments – the absorption spectra The absorption spectra: 1. Bacteriochlorophyll a 2. Chlorophyll a (primary pigment) 3. Chlorophyll b (accessory pigment – extends the usability of the spectrum. 4. Phytoerythrobilin 5. Beta-carotene Pigments – structure and function Chlorophyll a: o Green primary pigment (absorbs purple / red light). o Made up of an antenna complex that is associated to a photochemical reaction centre, forming a photosystem. Chlorophyll b: o Olive-green accessory pigment (absorbs blue / red light). o Similar structure as chlorophyll a. Carotenoids: o Yellow, orange, red, or brown accessory pigments. o Pass light energy absorbed to chlorophyll a. o Provide photoprotection (prevents oxidative damage during photosynthesis). Energy has three possible fates when hitting pigments: o Extra energy converted to heat and/or light. o Transferred to neighbouring chlorophyll molecules by resonance energy transfer. o Transferred from a negative charged high-energy electron to another nearby molecule (electron acceptor). When light hits the complex, electrons are excited and this energy is transferred to the reaction centre by resonance energy transfer. Light dependent reaction - photolysis The oxygen evolving complex catalyses the splitting of two water molecules into protons, oxygen, and electrons. 2H2O -(4 photons)-> 4H+ + O2 + 4e- The proton gradient drives the ATP synthase to generate ATP (photophosphorylation). Light dependent reaction – photophosphorylation Non-Cyclic Photophosphorylation (Main Pathway): Light Absorption: Photosystem II (PSII) absorbs light, exciting electrons in its chlorophyll molecules. Electron Transport: Excited electrons are passed to the primary electron acceptor and travel through the electron transport chain (ETC). Water Splitting: To replace lost electrons, water is split (photolysis), producing oxygen (O₂), protons (H⁺), and electrons. ATP Generation: As electrons move through the ETC, they drive the pumping of H⁺ ions into the thylakoid lumen, creating a proton gradient. H⁺ ions flow back into the stroma through ATP synthase, producing ATP. Photosystem I (PSI) Activation: Electrons reach PSI, which absorbs additional light energy to excite electrons to a higher energy level. NADPH Formation: High-energy electrons are transferred to NADP⁺, along with H⁺, to form NADPH. Cyclic Photophosphorylation (Supplementary Pathway): Electrons from PSI are cycled back to the ETC instead of reducing NADP⁺. This generates additional ATP but does not produce NADPH or oxygen. It helps balance the ATP/NADPH ratio for the Calvin cycle. Light-independent reaction – Calvin-Benson- Bassham cycle Location: occurs in the stroma of chloroplasts. Purpose: converts atmospheric CO₂ into organic molecules (e.g., glucose). Carbon Fixation: CO₂ combines with ribulose bisphosphate (RuBP) using the enzyme RuBisCO to form a 6- carbon compound. Reduction: The 6-carbon compound splits into two molecules of 3-phosphoglycerate (3-PGA). ATP and NADPH are used to convert 3-PGA into glyceraldehyde-3-phosphate (G3P), a 3-carbon sugar. Regeneration of RuBP: Some G3P molecules are used to regenerate RuBP, using ATP, allowing the cycle to continue. Outputs: 1 G3P molecule is exported to synthesize glucose and other carbohydrates. Requires 6 CO₂, 18 ATP, and 12 NADPH for 1 glucose molecule = 6 turns of the cycle (1 G3P made per CO2, 2 G3P is needed for glucose, however only 1 of 6 turns can be used because 5 are needed for RuBP regeneration, the other 1 is from natural accumulation IDK). Carbon concentration mechanisms Rubisco is an inefficient enzyme – it can fix oxygen rather than carbon dioxide (as a result of kinetic properties, concentration of CO2 and O2, and temperature) = photorespiration. C4 plants: o Bundle sheath cells are in close proximity with mesophyll cells. o CO2 is quickly converted and fixed to form a C4 acid and shuttled to bundle sheath cells. o The acid is decarboxylated and used in the calvin cycle / regenerated. o Higher concentration of CO2 in the bundle sheath cells prevents photorespiration. CAM plants: o Stomata in the leaves remain shut during the day. o CO2 is stored as malic acid in the vacuole. o During the day, malic acid is decarboxylated to release CO₂, which enters the Calvin cycle. o This temporal separation minimizes photorespiration and conserves water. Algae: o CO₂ or bicarbonate (HCO₃⁻) is actively transported into the cell and concentrated around Rubisco in specialized compartments called pyrenoids (within chloroplasts). o This ensures a high CO₂ concentration near Rubisco, enhancing carbon fixation efficiency. o Some algae can also utilize C4-like pathways or convert bicarbonate into CO₂ for fixation. Mitochondria – main features The number of mitochondria per cell varies according to the energy requirements of the cells. Membranes: Inner and outer membranes create an intermembrane space and the internal matrix. Inner membrane is a highly specialised lipid bilayer that is particularly impermeable to ions – site of the electron transport chain and ATP synthesis. Inner membrane is highly convoluted, forming cristae that increase the surface area. Internal structures: The matrix contains enzymes involved in the citric acid cycle. Electrons and protons Oxidation = removal of an electron. Reduction = addition of an electron. Hydrogen consists of an electron and a proton: H = e- + H+ Protons are not involved in redox reactions (do not reduce or oxidise) – if 2H is added to FAD, FADH2 is produced which is a reduced molecule. Glycolysis Step 1: Glucose is phosphorylated to glucose-6- phosphate (G6P) using ATP. Step 2: Molecules are rearranged and phosphorylated to form fructose-1,6-bisphosphate (F1,6BP) using another ATP. Step 3: F1,6BP is split into two 3-carbon molecules. Step 4: G3P is formed. Step 5: Each G3P is oxidized, and NAD⁺ is reduced to NADH. Step 6: ATP and pyruvate is formed as more reactions occur. Net Yield: ATP: 2 ATP (4 produced, 2 consumed) NADH: 2 NADH Pyruvate: 2 molecules per glucose molecule. Link reaction Transport of Pyruvate: pyruvate, produced in the cytoplasm during glycolysis, is transported into the mitochondrial matrix via a transport protein. Decarboxylation: pyruvate (3-carbon molecule) undergoes oxidative decarboxylation, where one carbon is removed as carbon dioxide (CO₂). NAD⁺ Reduction: the remaining 2-carbon molecule (acetyl group) is oxidized, and NAD⁺ is reduced to NADH. Formation of Acetyl-CoA: the acetyl group combines with coenzyme A (CoA) to form acetyl-CoA, which enters the citric acid cycle. Summary: Inputs: 1 pyruvate, NAD⁺, CoA. Outputs: 1 acetyl-CoA, 1 NADH, 1 CO₂ (per pyruvate). Krebs (citric acid) cycle. Oxidative phosphorylation Electron Transfer: o Electrons from NADH and FADH₂ (produced in earlier stages) are transferred to the ETC at Complex I (for NADH) and Complex II (for FADH₂). o Electrons are passed along the chain through a series of redox reactions, moving from one protein complex to another. o Ubiquinome shuttles electrons from Complex I (from NADH) or Complex II (from FADH₂) to Complex III. o Cytochrome c transfers electrons between Complex III and Complex IV, enabling oxygen to be the final electron acceptor. Proton Pumping: o As electrons move through Complex I, Complex III, and Complex IV, energy is released and used to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space, creating a proton gradient. Oxygen as the Final Electron Acceptor: o At Complex IV, electrons combine with oxygen (O₂) and protons to form water (H₂O). Oxygen is the terminal electron acceptor. ATP Synthesis: o The proton gradient created by the ETC generates a chemiosmotic potential (proton-motive force). o Protons flow back into the matrix through ATP synthase, a protein that uses this energy to phosphorylate ADP into ATP. Summary: Inputs: NADH, FADH₂, O₂, ADP, Pi. Outputs: ATP (approximately 28–34 per glucose), H₂O, NAD⁺, FAD. Chemiosmosis ATP synthase = multi-subunit protein. Chemiosmosis is the process by which the energy stored in the proton gradient is used to drive ATP synthesis. Chemiosmosis: the energy coupling mechanism: Proton Gradient Creation: o The electron transport chain (ETC) pumps protons from the mitochondrial matrix to the intermembrane space. o This creates: ▪ A chemical gradient (difference in proton concentration). ▪ An electrical gradient (charge difference across the membrane). Proton Flow: o Protons move back into the matrix through ATP synthase, down their electrochemical gradient. Energy Conversion: o The energy from proton flow is converted into mechanical energy (rotation of ATP synthase) and then into chemical energy (formation of ATP). Mitochondria uncoupling: o Uncoupling proteins dissipate the energy of substrate of oxidation as heat. o Allows continuous reoxidation of coenzymes that are essential to metabolic pathways. Compartmentalisation Compartmentalisation of cells increases the speed of reactions and allows increased diversity in the way cells are organised. Mitochondria = many grouped around the nucleus to provide energy for protein synthesis. Endosomes = vesicles formed when a cell takes in an outside substance. Lysomes = degrade the contents of endosomes. Autophagosomes = digest parts of the cell, recycling within the cell. Peroxisomes = metabolic reaction (e.g., lipid breakdown). Compartmentalisation (percentage of membrane) strongly relates to function – greater percentage of inner membrane of mitochondria relates to higher metabolic activity, greater percentage of RER and Golgi membrane relates to greater protein production, etc. Membrane structure and function Function: Controlling transport. Concentrating enzyme activity. Controlling cell communication. Connecting cells. Recognising cells. Structure: Fluid-mosaic mode. Most membranes 5-10nm thick compared to typical 20,000nm wide cells. Bacteria possess membranes with a different composition to animal cell membranes. Enveloped viruses are enclosed by membranes, usually derived from the host cell (enveloping occurs when exiting cells, aiding fusing to the next). Membrane lipids - phospholipids Phosphoglycerides = glycerol based. Sphingolipids = sphingosine based. There is a lot of variation in phosphorlipids due to different combinations of head groups, carbon chains length, and saturation states. Passive transport across membranes Hydrophobic (nonpolar) molecules can dissolve in the lipid bilayer and pass through the membrane rapidly through simple diffusion. Facilitated diffusion: o Channel proteins: ▪ Can be open and closed. ▪ Specific to certain molecules to regulate transport. o Carrier proteins: ▪ Molecules too big for channels are transported (the bigger the channel, the less transport control there is so carriers provide a transport mechanism with greater regulation). ▪ Will be open on one side of the membrane and closed on the other side. Active transport Cells contain many solutes, and therefore water will move into the cell by osmosis and burst the cell if unregulated: Cells control intracellular osmolarity by activiely pumping out inorganic ions so that their cytoplasm contains a lower concentration of inorganic ions than the extracellular fluid. Sodium potassium pump example: o 3 Na+ pumped out. o Changes shape of the pump. o Energy is needed as this is against the gradient. o 2 K+ pumped in. o Changes shape of the pump. o Doesn’t require energy as this is down the gradient. Endocytosis Receptor-mediated endocytosis: Phagocytosis: Pinocytosis: Exocytosis Secreted proteins are transported to the plasma membrane in veiscles, when then fuse with the plasma membrane releasing their contents outside the cell. Proteins may be soluble inside the vesicle or embedded in the vesicle membrane. Constitutive secretory pathway: o Substances that need to be made and excreted constantly. o Unregulated membrane fusion. Regulated secretory pathway: o Secretion depends on signals. o Signal results in regualted membrane fusion for responsive secretion. Signal sequences The amino acid sequence of a protein can contain instructions (signal sequences) for where a protein belongs in a cell. Proteins can have multiple signal sequences – different ones are visible when the protein is folded and modified. Signal sequences are made up of specific combinations of amino acids and spaces between these combinations (that can be any amino acid). Transporting proteins within cells – gated transport The nuclear envelope is composed of two concentric membranes which are perforated by nuclear pores. Proteins are imported into the nucleus and mRNA is exported out of the nucleus through nuclear pores. Nuclear pore complex (NPC): Gatekeeping proteins read and check for nuclear import / export sequences (recognise nuclear localisation signals on proteins). Can transport in both directions at the same time. Transporting proteins within cells – transmembrane transport Pores in mitochondrial membranes would allow protons to pass through and therefore chemiosmotic gradients could not be maintained. Proteins with a specific signal sequence bind to inactive protein translocators, activating them. The signal sequence is often cut off by signal peptidase. The mature soluble protein is released into the organelle lumen. Transporting proteins within cells – motor proteins Three types of motor protein: Myosin: o Transports along actin filaments. Kinesin: o Transport along microtubules. o - -> + (towards the plasma membrane). o Every 'step' uses 1 ATP Dynein: o Transport along microtubules. o + -> - (towards the nucleus). Types of information received Types of stimuli cells can respond to include: Growth factors Death signals Cell-cell contact Cell-extracellular matric contact Hormones Light (photons)] Odorants Touch (mechanical stimulus) Temperature Oxygen levels PH Magnetic fields Mechanisms of communication - overview Direct cell-cell signalling. Signaling by secreted molecules: o Endocrine signaling (hormones travel through the bloodstream to target cells). o Pancrine signaling (medium-distance, within the general region). o Autocrine signling (cell sends to itself). Most cell communication involves activation of a specific receptor protein (typically in the plasma membrane) by a specific signaling molecule (ligand) - receptor activation results in transmission of signal into the cell through signal transduction, leading to a cellular response. Signal reception Types of receptor, including: Ligand-gated ion channels = channel only opens when signal is received, three types. G-protein coupled receptor (GPCR) = Enzyme-linked receptor = enzyme activated when signal received. Receptor activation: Ligand binds, resulting in activation and therefore conformational change of receptor. In enzyme-linked receptors, this leads to the activation of enzymes that relay the signal into the cell / the receptor itself is an enzyme. o Enzyme-linked receptors are also known as tyrosine kinase receptors. o The enzyme activity on one receptor helps to phosphorylate and activate the other receptor molecule in the dimer. o This change acts as a signal for other enzymes to be recruited and therefore the signal can be relayed and passed further into the cell. Signal transduction – second messengers Signal amplification via second messengers allows a weak signal to be amplified so that cells can respond rapidly. Examples of second messengers: o Cyclic AMP o Cyclic GMP o Inositol triphosphate o Diacylglycerol o Calcium Example – adrenaline and second messengers: o Adrenaline binds to a beta-adrenergic receptor in the cell membrane. o This results in the G-protein complex dissociating and activating adenylyl cyclase. o Adenylyl cyclase catalyses the activation of the second messenger cAMP using ATP. o This leads to a second messenger cascade. Signal transduction – protein phosphorylation Kinases = class of enzymes that attach phosphate groups to a target protein. Phosphorylation of the target protein changes the protein structure – conformational change (because phosphate is negatively charged) - leading to a change in activity. Phosphatases = class of enzymes that reverse phosphorylation by removing the phosphate group. Protein phosphorylation is an example of post-translational modification o The ability of proteins to have different functions means greater possibilities for cells. Olfactory sensing Olfactory receptors bind to volatile odour molecules. Individual odorant molecules can bind to many different receptors, but with different affinities – smells are made up of a number of odorant molecules triggering a pattern of receptor activation which is then interpreted by the brain. Light sensing Dealing with multiple signals Differentiation Differentiation = turning an undifferentiated, generalist cell into a differentiation, specialised cell. Stem cells: o Cell division in stem cells is asymmetric – producing two different daughter cells: ▪ One daughter cell is a stem cell (self renewal). ▪ The other becomes differentiated. o Totipotent = only present in the early stage following fertilisation, capable of producing any cell type. o Pluripotent = capable of producing any cell within a major lineage (e.g., ectoderm, endoderm, mesoderm). o Multipotent = capable of producing a restricted set of related cells (e.g., haemopoietic cells). Triggering differentiation: o Stem cells receive differentiation signals that bind to receptors, transmitting signals into the cell leading to changes in gene expression. o Different combination of signals = different specialisation. o Same combination of signals may occur, however different amounts of the signals = different specialisation. As cells become differentiated, more and more genes become silenced (from euchromatin – heterochromatin) and only those needed to carry out the cell's specialist functions remain accessible in euchromatin. Epigenetics and differentiation The switch between euchromatin and heterochromatin is controlled by enzymes that can attach to methyl / acetyl groups either directly to DNA or to histone proteins that bind to DNA. DNA methylation = silences genes. Histone acetylation = leads to euchromatin regions and increased gene expression. Histone methylation = can either increase or decrease gene expression depending on which pats of the histone protein are modified. The cell cycle Single-celled organisms = cell cycle can repeat continuously as long as there is sufficient space and nutrients. Multi-cellular organisms = cell cycle is more strictly controlled. Cells with slow cell cycles typically exist in G0. Cells that never divide (fully differentiated) are called post-mitotic cells. Due to ageing some cells lose the ability to divide (not originally supposed to be post-mitotic) – senescent cells. G1 phase Cell undergoes considerable protein synthesis. Cell duplicates organelles. Cells are highly metabolically active at this stage and require lots of energy. S phase Cell has grown big enough to duplicate its DNA. An extra copy of each chromosome is made and the two copies are joined at the centromere. G2 phase Second rapid period of cell growth (readying for mitosis). DNA checks to see whether it has been copied correctly. Many cancer cells go straight from S phase to mitosis. M phase Nuclear envelope breaks down, mitotic spindle forms, and the chromosomes are separated – cell splits in two (cytokinesis). Several stages in M phase: o Prophase o Prometaphase o Metaphase o Anaphase o Telophase Kinetochore = protein complex that attaches chromosomes to microtubules, leading to the segregation of the chromosomes during mitosis. Cytokinesis = actin filaments align around the middle of the cell, forming a cleavage furrow and eventually splitting the cell. Cell cycle regulation There are critical stages during the cell cycle called commitment points when the cell has to make a decision about whether to proceed to the next stage based on the environmental conditions. Once DNA has been copied, the cell must either complete the cycle or die – a cell with 2x the amount of chromosomes is not viable. Checkpoints: o End of G1 = DNA damage checkpoint, entrance to S is blocked if genome is damaged. o S = DNA damage checkpoint, DNA replication halted if genome is damaged. o End of G2 = entrance to M is blocked if DNA replication is not complete. o Metaphase part of M = anaphase is blocked if chromatids are not properly assembled on the mitotic spindle. Senescence The Hayflick limit = most cells from multicellular organisms have a limited lifespan, typically around 40-60 cell divisions (the limit). Cells that are not actively dividing are in the G0 phase of the cell cycle. Senescent cells are permanently stuck in the G0 phase and cannot enter the cell cycle. To reduce the risk of developing cancer (lose control of our own cells and they proliferate uncontrollably), cells have evolved built-in limits on cell division. Telomere shortening following cell division limits the number of times a cell divides – cancer cells rebuild their telomeres using telomerase. HeLa cells: o Type of cancer cell. o Isolated from a cervical tumour – found to be immortal and could be cultured indefinitely. o Used to create the first human cell line, used to test the first polio vaccine. o Immortal cell lines can now be generated by transforming normal cells with viral genes.  Apoptosis vs necrosis Necrosis = uncontrolled cell death, associated with disease. Apoptosis = programmed cell death / suicide, essential part of normal health and development. Necrosis Apoptosis Morphological features: Morphological features: - Loss of membrane integrity. - Membrane blebbing, but no loss of integrity. - Ends with total cell lysis. - Ends with fragmentation of cell into smaller bodies. Biochemical features; Biochemical features: - No energy requirement (passive). - Tightly regulated process involving activation and enzymatic steps using ATP. Physiological significance: Physiological significance: - Affects groups of cells. - Affects individual cells. - Significant inflammatory response. - No inflammatory response. Importance of apoptosis Embryo development – sculpting tissue, quicker and easier to control than making specific structures initially. Immune system – destroying self-reacting immune cells (B and T cell receptors are randomly made and sometimes are accidentally self-reactive, therefore need to be killed), as well as virus infected cells. Homeostasis = counter-balance to cell division, removes old / damaged cells. Cancer = radiotherapy and most chemotherapy drugs work by inducing apoptosis. Triggering apoptosis - overview Two main pathways for triggering apoptosis: Receptor mediated (extrinsic pathway) o 3 possible types of death-inducing ligands can bind to death receptors. Mitochondria mediated (intrinsic pathway) Caspases: Family of 12 proteases that exist as inactive pro-enzymes in cells – following activation by cleavage they can activate other caspases in a cascade. Two types of apoptotic caspases: o Initiator caspases = activate other caspases. o Effector (executioner) caspases = break down cellular components such as the cytoskeleton and DNA. Triggering apoptosis – DISC assembly DISC = death-inducing signaling complex Ligand binds to receptor Causes trimerisation Platform inside membrane is formed Procaspase 8 is able to bind and cap removed (activated) Activa caspase 8 can then activate procaspase 3 Continues to activate other executioner caspases to bring about apoptosis Role of mitochondria in apoptosis Cytochrome is located in the inner mitochondrial membrane and is an essential component of the electron transport chain. To trigger apoptosis, pores form in the outer mitochondrial membrane – this allows the release of cytochrome C from the cytosol. Cytochrome C binds to other cytosolic proteins to form a multi-protein complex = apoptosome: o Formation requires cytochrome C, protein called Apaf-1, procaspase 9, and ATP. o End result is the cleavage and therefore activation of procaspase 9 into active caspase 9 (initiator caspase). Control of cytochrome C release: o Pro-apoptotic members of the Bcl-2 family (proteins) insert themselves into the mitochondrial surface and promote the formation of large pores = allow cytochrome C release. o Anti-apoptotic members exist in the mitochondrial outer membrane and block the action of the pro-apoptotic members = prevent cytochrome C release. o The balance between the two determines how difficult it is to induce apoptosis. Forming tissues introduction Human bodies are composed of approximately ten trillion cells and over 200 specialised cells. Cells must be connected strongly together and work in a coordinated fashion in order for tissues and organs to function properly. Infastructure holding cells and tissues together must be malleable enough to cope with stress, changing environments, and to allow repairs. Cell junctions – gap junctions Gap junctions = channel proteins that align between two cells. Directly connect the cytoplasms of the two cells and allow direct communication between them. Gap junctions do not connect to the cytoskeleton. Main functions: o Exchange of metabolites. o Pasage of communication signals. Cell junctions – tight junctions Tight junctions = produce a virtually impermeable barrier between cells, which are connected by cell surface proteins. Cell surface proteins – recognise the same molecule on neighbouring cell, forming a tight bond: o Claudins o Occludins Cell surface proteins are connected to the actin cytoskeleton to provide strength. Anchor proteins = called the ZO complex, connect the clauding and occluding molecules to actin filaments. Cell junctions – adhering junctions Cadherins = cells surface molecules that allow similar cells to recognise each other and form connections. o Interaction between two identical cadherin molecules on different cells leads to the formation of a junction between them. Desmosomes = connect cells through their intermediate filaments. o Anchor proteins are needed to connect the caherin molecules to the cytoskeleton – these proteins are large and therefore the gap between cells is wider than tight junctions. Adherens junctions = connect to actin filaments. o Also use cadherin mloecules and anchor proteins. The extracellular matrix - overview ECM = scaffolding system surrounding cells that they attach to, consisting of fibres and large proteins such as collagen. The ECM is made by cells. Cells attach to the ECM through integrins (cell surface molecules). ECM provides structure and both mechanical and biochemical support for tissues. Basement membrane = formed by thin sheets of ECM at the base of tissues such as skin. Major components of the ECM: o Collagen o Fibonectin (helps to strengthen the connection between cell integrins and fibres in the ECM). o Proteoglycans (large sugar-coated proteins). The extracellular matrix - collagen Most abundant protein in the body - major structural protein, forming molecular cables that strengthen the tendons, bones and teeth are made by adding mineral crystals to collagen. Collagen provides structure to the body, protecting and supporting softer tissues and connecting them with the skeleton. Structure = composed of chains, wound together in a tight triple helix. At least 28 types of collagen (example of redundancy – back up system, if there was only one type and the gene was mutated, the body would likely not survive), but most tissues are mixtures of 5 main types: o Type I = 90% of collagen, densely packed, used to provide structure to the skin, bones, tendons, and ligaments o Type II = found in elastic cartilage, provides joint support. o Type III = found in muscles, arteries, and organs. o Type IV = found in basement membranes. o Type V = found in the cornea, some layers of skin, hair, and placenta tissue. The extracellular matrix - proteoglycans Proteoglycans = proteins that are heavily glycoslated, contain very long chains of sugar molecules branching off of the main protein chain. At least 43 different types: o Chondroitin sulfate chains = good at absorbing compressing forces. o Hyaluronan (hyaluronic acid) = lubrication, very common in the brain. Proteoglycans function to lubricate the ECM, and also modify the activity and stability of the other proteins in the ECM. The extracellular matrix - fibronectin Fibronectin = glycoprotein that has binding sites for multiple ECM proteins (such as collagen and proteoglycans) as well as binding sites for cell surface integrins The extracellular matrix - integrins Integrins = cell surface molecules that act as matrix receptors. Functions: o Connect cells to the ECM. o Transmit signals into the cell, allowing it to sense and respond to its environment. Structure: o Consist of an alpha and beta chain. o Different combinations of these chains allow them to recognise a range of ECM components. o Change into an open conformation when binding to the ECM. o Cytoplasmic tails are connected to the actin cytoskeleton throguh the use of anchor/adaptor proteins = adhesion complex. The extracellular matrix - hemidesmosomes Hemidesmosomes = can be used to attach cells to basement membranes. Connect the ECM to intermediate filaments – link goes through integrin molecules. Provide additional mechanical strength to cells and tissues. The extracellular matrix - healing Fibroblasts = most important type of cell in maintaining the ECM. o Become activated in response to injury and secrete collagen to close the wound as quickly as possible – often leading to scar formation (fibrotic tissue). Evolution of multicellularity Multicellularity probably independently evolved at least 25 times. o Advantages: o Improved acquisition of resources. o Increased resistance to physical/chemical stress. o Protection from predation (harder to be engulded by unicellular predators). o More efficient colonisation of new territories. o Opportunity for cell differentiation and therefore specialisation among different cell types. Disadvantages: o Energetic costs from the synthesis of adhesion and communication molecules (quarter of all proteins is collagen – consequence of multicellularity). o Physical limitations from reduced freedom or movement/proliferation. o Vulnerable to exploitation. However, advantagegs outweigh disadvantages. Examples of single celled organisms that can form multicellular aggregates (non-clonal): o Bacterial biofilms. o Social amobae / slime moulds. o Algae. Clonal multicellularity = all of the cells in an organism have the same DNA which is associated with animals, plants, and fungi – likely to have emerged around 800 million years ago but is not understood. Bacterial biofilms Multicellular aggregates of prokaryote cells, both bacteria and archaea. Cells attach to a surface and are joined by other cells to form microcolonies – can expand to form biofilms. Release individual cells for dispersal. Cells secrete proteins, forming something similar to the ECM. Social amoeba Dictyostelium = amoeba that can alternate between being single-celled and multicellular. Life cycle: o Spore o Single-celled amoeba o Mitosis o Aggregating amoeba o Elongating mound o Mound falls over – forms a grex o Sporing body o Cycle repeats Social amoeba used to be called slime moulds. When food is scarce, the cells begin to aggregate to aid dispersal. Algae Chlamydomonas species = unicellular algae that have flagella to help them swim but repurpose the flagella during cell division. Volvox species = multicellular algae that form colonies of thousands of cells, including specialised, germ, and somatic cells. Robustness - overview Robustness = the ability of a cell to maintain performance and function in the face of internal external pertubations. Redundancy = many genes and aspects of cells and organisms are replicated and have 'back ups', which are redundant when everything is healthy and operating correctly, but aid survival when there are abnormalities (e.g. many types of collagen, although type I accounts for 90% of collagen, if this is not made/incorrect then it can be substituted for by the previously 'redundant' types). Robustness – high temperatures Fever during infection can help the immune response by: o Increased movement of white blood cells. o Increased proliferation of white blood cells. o Enhanced rate of phagocytosis. However: o Most enzymes are optimised to work at 37C. o The cell membrane becomes much more fluid. o Proteins don’t fold properly. How membranes respond: Composition: o More saturated fatty acids. o More cholersterol. Heat shock proteins: o Proteins produced when a cell is exposed to increased temperature. o Increase in expression in response to other stresses (e.g., O2 deprivation, nutrient deprivation). o Main function is to help proteins fold properly so they can function properly. o Many copies as they are very important (redundancy). Robustness – extreme heat Geogemma barossii – archaea found found growing in a hydrothermal vent, can survive at 130C. Adaptations: High GC levels in third codon position = stronger bonding. Supercoiling DNA = harder to separate. Temperature stable proteins. Changes to membrane composition, including ether rather than ester bonds = stronger bonding between lipids. Robustness – low temperatures Problems: o Slow enzyme reactions. o Rigid and viscous membranes. o Ice crystals (can puncture holes in cell membranes). Glycoproteins = attached sugars function to stabilise the proteins structure in the extracellular environment and therefore offer some protection. o Antifreeze glycoproteins used in blood cells of antarctic fish stop/organise ice crystal formation Glycolipids = similar protective functions to protect from stress. Robustness – low oxygen Anoxia (no O2) --> hypoxia (low O2) --> normoxia (normal O2) --> hyeroxia (high O2). General hypoxia can occur: o Environmental O2 levels are low (e.g. high altitude). o Premature babies. o Poisoning. o Anaemia. Local hypoxia can occur because of: o Ischaemia = blood supply is cut off following heart attacks and strokes. o Cancer = tumour growth collapses blood vessels. Hypoxia inducible factors (HIFs) = oxygen sensing mechanisms that move to the nucleus and alter gene expresssion in response to hypoxia – normally degraded by the proteasome when oxygen is present. o Cell cycle stops (arrested at G1). o Switches to glycolysis and anaerobic metabolism to produce ATP. o Rapid down-regulation of protein synthesis. Angiogenesis = making new blood vessels, response to hypoxia as growth factors stumulate blood vessels to branch off, proliferate, and move towards the hypoxic cell. Robustness – nutrient deprivation Quiescence = state of reversible cell cycle arrest. Autophagy = cells form double-membraned vesiclse (autophagosomes) that sequester organelles/proteins/cytoplasm for delivery to the lysosome where they can be recycled to maintain nutrient and energy homeostasis. Proteasomes = multi-protein complexes that are able to degrade unwanted/damaged proteins to recycle amino acids for more protein synthesis. o Operate on one protein at a time for more precision. o Degrade proteins damaged by shock and deprivation. Robustness – toxic environments Toxic environments: o Radiation. o Free radicals (O2 molecule with unpaired electron = highly reactive). o Oxidative stress. o Toxins. o Poisons. Reative oxygen species: o O2 is the final electron acceptor in aerobic respiration, accepting 4 electrons, but sometimes this goes wrong and a highly reactive oxygen species is created. o Glutathione = major anti-oxidant that readily donates electrons to ROSs to form more stable molecules. o Superoxide dismutases = enzymes that convert highly reactive superoxide free radical (O2-_ to less reactive hydrogen peroxide (H202). o Catalase = then converts hydrogen peroxide (still a ROS) to water and oxygen. Robustness – repairing DNA damage DNA damage: Damage detected. Cell cycle stops. DNA repair is initiated. Too extensive = apoptosis. Unicellular life has a higher tolerance for DNA damage. Cancer = disease characterised by a loss of control over our own cells, happens when genetic damage occurs in cells that is not correctly repaired. Some mutations can change the way cells respond to signals, meaninf that these cells proliferate when they shouldn’t. ONE OF THE QUESTIONS How do heat shock proteins protect cells from high temperatures: a) Assist protein folding b) Decrease membrane fluidity c) Increase membrane cholesterol d) Reduce mitochondrial permeability e) Increase DNA supercoiling  M C 1 2 10000 bp 3000 bp 2000 bp 1500 bp 1000 bp 750 bp 500 bp 250 bp 240 bp