Cell Structure and Function

1. all living organisms are composed of one or more cells

2. the cell is the basic unit of structure and organisation

3. all cells arise only from pre-existing cells

• universal similarities bewteen cells:

  1. DNA as the heritable material, RNA as a messenger and proteins as the workers

  2. major cellular organelles - functions and arrangements within the cell

  3. ATP as an energy source

• the central dogma:

  • DNA → RNA → PROTEIN

what is cell theory

• both have:

  • plasma membrane

  • cytosol

  • DNA

  • RNA

  • protein and ribosomes

• eukaryotic cells have membrane-bound organelles and are much larger

• prokaryote cells lack a membrane-bound nucleus

describe the similarities and differences between prokaryotes and eukaryotes

• the cytoplasm is everything inside the plasma membrane except the nucleus

• the fluid portion of the cytoplasm is the cytosol

  • water plus dissolved and suspended substances (e.g. ions, ATP, proteins, lipids)

• major organelles include:

  • nucleus

  • endoplasmic reticulum (smooth and rough)

  • golgi apparatus

  • vesicles

    • these four make up the endomembrane system (along with plasma membrane, they work together to package, label and ship molecules)

  • mitochondria

  • ribosomes

what is the cytoplasm

• the plasma membrane is a selectively permeable barrier controlling the passage of substances in and out of the cell

• made up of a double layer of phospholipids with embedded proteins:

  • hydrophilic polar heads (phosphate)

  • hydrophobic lipid tails (fatty acids)

    • arranged as a double layer, tail to tail

    • much of our body is hydrophobic or ‘water loving’

    • fats are hydrophobic (‘water hating’)

    • fats in cell membrane provide a barrier to water

describe the plasma membrane

• membrane proteins mediate movement of hydrophilic substances

• are often amphipathic, meaning they have both hydrophilic and hydrophobic regions

• integral proteins:

  • embedded (partially or fully) into the membrane

    • e.g. transmembrane proteins are integral membrane proteins that fully span the entire membrane, contracting both extracellular and cytoplasmic areas

• peripheral membrane proteins:

  • are associated with the membrane, but not actually embedded within it

describe the plasma membrane proteins

• transport

  • e.g. channels, transporters

  • may be general or selective, gated or not

• enzymatic activity

  • carry out chemical reaction, may or may not be a part of a team of enzymes

• signal transduction

  • external signaling molecule causing communication of information to the inside of the cell

• cell-cell recognition

  • use of glycoproteins (carbohydrate + protein) as molecular signature of the extracellular side of the cell

• intercellular joining

  • e.g. gap junctions or tight junctions

• attachment to the cytoskeleton and extracellular matrix (ECM)

  • e.g. fibronectin mediates contact between cell surface integrins and ECM (e.g. collagen)

  • can facilitate movement

what do the plasma membrane proteins do

• membranes are not static

• the membrane is a mosaic of molecules bobbing in a fluid bilayer of phospholipids

• cell specific and dynamic repertoire of membrane-bound proteins present as required

describe the movement of membranes

• largest distinct structure inside the cell

• enclosed by double lipid bilayer called nuclear envelope, continuous with rough ER

• entry and exit through nuclear pores

• nucleolus: rRNA production, assembly of small and large subunits of ribosomes

• functions:

  • to house/protect DNA

  • make RNA

  • pores regulate movement of substances (e.g. protein and mRNA) in and out

  • molecule segregation to allow temporal and spatial control of cell function

describe the nucleus

• DNA wrapped 2x around group of 8 histones, to form nucleosomes - collectively known as chromatin

• as the cell prepares for cell division, chromatin condenses to form chromatin fibres then condenses further into loops and then stacks as fully condensed chromosomes

• most of the time, our DNA is present in our cells as chromatin and chromatin fibres

• chromosome — comprises many genes, usually >1000

• gene — a DNA segment that contributes to a phenotype/function

describe deoxyribonucleic acid (DNA) in the nucleus

• two subunits, small and large made of ribosomal RNA (rRNA) in complex with many proteins

• rRNA is made in the nucleolus

• subunits assemble in the nucleolus and leave through nuclear pores

• function: protein production (translation), found in two places within the cell:

  • free in the cytoplasm — making proteins to be used in cytosol (non-endomembrane destinations)

  • attached to the RER — making non-cytosolic proteins/endomembrane

describe ribosomes

• the ER is an extensive network of tubes and tubules, stretching out from the nuclear membrane

• two types: rough ER and smooth ER

outline the endoplasmic reticulum

• continuous with nuclear envelope dotted with attached ribosomes

• proteins enter lumen within the rough ER for folding

• rough ER membrane surrounds the protein to form transport vesicles destined for the Golgi

• major function is production of:

  • secreted proteins

  • membrane proteins

  • organelle proteins

describe the rough endoplasmic reticulum

• extends from the rough ER

• lacks ribosomes: doesn’t make proteins

• synthesises lipids, including steroids and phospholipids

• stores cell-specific molecules

• functions of smooth ER vary greatly from cell to cell

  • very cell/tissue-type specific

• examples:

  • liver: houses enzymes for detoxification and for glucose release

  • muscle: calcium ions

describe the smooth endoplasmic reticulum

• the ‘warehouse’ of the cell

• this complex is made up of 3 to 20 flattened membranous sacs called cisternae, stacked on top of one another (like ‘pita bread’)

• functions:

  • modify, sort, package, and transport proteins received from the rough ER using enzymes in each cisternae

• formation of:

  • secretory vesicles (proteins for exocytosis)

  • membrane vesicles (PM molecules)

  • transport vesicles (molecules to lysosome)

describe the Golgi apparatus - receiving and modifying

• each sac or cisternae contains enzymes of different functions

• proteins move cis to trans from sac to sac

• mature at the exit cisternae

• travel to destination within vesicles

• modifications occur within each sac (formation of glycoproteins, glycolipids, and lipoproteins)

describe the Golgi apparatus: to destination

• main function: generation of ATP through cellular respiration

• mitochondria are made up of:

  • outer mitochondrial membrane

  • inner mitochondrial membrane, with folds called cristae f

  • fluid filled interior cavity, called the mitochondrial matrix

• despite all of these membranes, mitochondria are not part of the endomembrane system

• the more energy a cell requires, the more ATP it must take, and the greater the number of mitochondria present

• mitochondria carry a separate small (37 genes) genome encoding mitochondrial-specific products

describe the mitochondria

• structural support system of the cell

• fibres or filaments that help to maintain the size, shape, and integrity of the cell:

  • act as scaffolding across the cell

  • involving in intracellular transportation and cell movement

• three types of fibres (from smallest to largest):

  • microfilaments

  • intermediate filaments

  • microtubules

describe the cytoskeleton

• diameter: ~7nm

  • comprised of actin molecules assembled in two long chains, twisted around each other

  • found around the periphery and lining the interior of cell

• function:

  • bear tension and weight by anchoring cytoskeleton to plasma membrane proteins, and promote amoeboid mobility if required (e.g. macrophage)

  • assembled and disassembled as required — they are dynamic

describe the microfilaments in the cytoskeleton

• diameter: 8-12nm

• comprised of diverse range of different materials; one example: keratin

• found in the cytoplasm of the cell

• function:

  • bear tension and weight throughout cell, e.g. during cell anchoring

  • acts as a scaffold for cellular organelles, e.g. the nucleus

• usually the most permanent of cytoskeletal structures — they are less dynamic

describe the intermediate filaments of the cytoskeleton

• diameter: tubular structure, 25nm with central lumen of 15nm diameter

• comprised of tubulin dimers (alpha and beta), coiled, to form a tube

• extends from centriole into cytoplasm/nucleus

• functions:

  • support cell shape and size

  • guide for movement of organelles

    • e.g. vesicles from Golgi to membrane

  • chromosome organisation — cell division

  • support and movement of cilia/flagella

• assembled and disassembled as required — are dynamic

describe the microtubules in the cytoskeleton

• ATP powers cellular work - it is our energy currency

• the hydrolysis of ATP to ADP and inorganic phosphate releases energy

outline the mitochondria as the ATP factory

• ATP cycle: the transfer of energy between complex and simple molecules in the body, with ATP as the mediator

• many cellular processes require energy in the form of ATP — they are not spontaneous

• simple molecules such as glucose, amino acids, glycerol, and fatty acids → anabolic reactions transfer energy from ATP to complex molecules → complex molecules such as glycogen, proteins, and triglycerides → catabolic reactions transfer energy from complex molecules to ATP

describe the ATP Cycle

• our major categories of fuel:

  • carbohydrates: broken down to simple sugars

  • proteins: broken down to amino acids

  • fats: broken down to simple fats

    • which are then absorbed

how is fuel needed to generate ATP

• glucose in food/intestines → glucose in bloodstream ← storage for harder times (facilitated by glucagon)

• glucose in bloodstream → into a cell (faciliated by insulin)

• cellular respiration ← cell → storage for harder times (glucose cross-linked together, called glycogen, in liver and skeletal muscle)

• cellular respiration → cellular work

describe the use of glucose in different parts of the body as it moves around

• the controlled release of energy from organic compounds to produce ATP

• conversion of glucose to ATP is due to 4 main steps:

  1. glycolysis

  2. pyruvate oxidation

  3. citric acid cycle (or Krebs cycle)

  4. oxidative phosphorylation

• the simplest overview: C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6 H₂O + Energy

outline the process of cellular respiration

• glycolysis (glucose → pyruvate) → cytosol

• pyruvate oxidation and Kreb’s cycle (acetyl CoA)→ mitochondrial matrix

• oxidative phsophorylation (electron transport and chemiosmosis) → across inner membrane

where does cellular respiration occur

• glycolysis invests and produces ATP

• occurs in the cytosol and oxygen is not required

• two ATP are invested

• the lysis of glucose to produces two pyruvate molecules

• four ATP are produced

• two ATP and 2NADH are produced (net)

• NADH is an electron carrier later in the electron transport chain

describe step 1 of cellular respiration: glycolysis

• pyruvate oxidation to form acetyl CoA

• this step links glycolysis to the citric acid cycle

• occurs in the mitochondrial matrix if oxygen is present

• produces no ATP, but produces 1NADH per pyruvate (or 2 per glucose) plus 1 CO₂

• the 2 carbon acetyl CoA molecule is able to enter the nitric acid cycle

describe step 2 of cellular respiration: pyruvate oxidation

• occurs in the mitochondrial matrix if oxygen is present

• results in (per glucose molecule):

  • 2 ATP

  • 6 NADH

  • 2 FADH₂

  • 4 CO₂

• requires oxygen — it is an aerobic process

• FADH₂ and NADH are electron donors in the electron transport chain

describe step 3 of cellular respiration: citric acid/Kreb’s cycle

• citrate → fatty acid synthesis

• α-Keto-glutarate → amino acid synthesis and neurotransmitter

• oxaloacetate → amino acid synthesis

• malate → gluconeogenesis

a series of reactions: product of one reaction is the substrate for the next

the citric acid cycle completes the extraction of energy from glucose

outline the citric acid cycle intermediates are used in other metabolic pathways

ATP genereated by direct transfer (from a substrate) of a phosphate group to ADP via substrate phosphorylation

what is substrate phosphorylation

ATP is generated from the oxidation of NADH and FADH₂ and the subsequent transfer of electrons and pumping of proteins

what is oxidative phosphorylation

• the electron transport chain

  • occurs at proteins within the inner membrane

  • requires oxygen — it is an aerobic process

  • NADH and FADH₂ are oxidised to donate electrons

  • electrons transfer from protein-to-protein along the chain in a series of redox reactions

  • at each transfer, each electron gives up a small amount of energy which enables H+ ions to be pumped into the intermembrane space

  • oxygen ‘pulls’ the electrons down the chain, and is then the final electron acceptor where it is reduced to water

  • NADH and FADH₂ from earlier steps are used here

• chemiosmosis

  • the hydrogen ions in the intermembrane space rush down their concentration gradient (chemiosmosis) through ATP synthase

  • this causes the ‘turbine’ within ATP synthase to turn

  • the rotation of the ATP synthase turbine enables the phosphorylation of ADP to generate ATP

  • this results in the production of 26 or 28 ATP (per glucose)

• ETC and chemiosmosis = oxidative phosphorylation

  • this is much more efficient than substrate phosphorylation

  • the bulk of ATP production occurs here

  • ‘fall’ of electrons down the chain enables movement of H+ ions into intermembrane spcae and generates a proton gradient which ‘drives’ the ATP synthase turbine

step 4: oxidative phosphorylation: the electron transport chain and chemiosmosis

• we can derive energy from more than just glucose

• fats, proteins, and more complex carbohydrates generate ATP also

• monomers enter glycolysis and the citric acid cycle at different points

outline how cellular respiration is versatile

• phosphofructokinase is the ‘gate-keeper’ for glycolysis; it catalyses step 3 — where glycolysis becomes irreversible

• inhibited by citrate and ATP

  • i.e. products of cellular respiration

• stimulated by AMP

  • AMP accumulates when ATP is being used rapidly

how is cellular respiration controlled

• insulin:

  • produced by beta cells of islets and langerhans in pancreas

  • function: promote glucose uptake into cells (for ATP production or storage in liver)

• glucagon:

  • prodcued by alpha cells of Islets of Langerhans in pancreas

  • function: stimulates the breakdown of glycogen to increase blood sugar levels

outline insulin and glucagon

• no glucose in cells

• no ATP from glucose

• no glycogen stored for harder times

• diabetes mellitus:

  • the ability to produce or respond to hormone insulin is impaired

  • results in abnormal metabolism of carhydrates and elevated levels of glucose in the blood

what happens if you lose the function of insulin

• type 1 or insulin-dependent diabetes:

  • body does not produce insulin, as beta cells of pancreas are destroyed, often this is autoimmune, or genetic or through environmental factors

  • affects 5-10% of diabetics, and onset usuaully occurs in children or adolescents

  • requires insulin replacement

• type 2 or non-insulin-dependent diabetes:

  • body produces insulin, but receptors are non function (insulin resistance)

  • most (>90%) diabetics are type II, usually adults over the age of 40

  • can be linked to other pathologies and obesity

outline diabetes mellitus

• diabetes mellitus is caused by a lack of functional insulin

• as a result, levels of glucose in the blood build up, well beyond normal homeostatic limits

• increased blood glucose alters the volume and osmolarity of blood, with subsequent pathological consequences

• two of the symptoms of this diseases are:

  • significantly increased hunger

  • significant weight loss

• these two symptoms seem to be in opposition to each other: if the patient is constantly hungry and eating, why would they then lose weight?

what are contradictory symptoms of diabetes mellitus

• cells need to be able to respond as a cell, and as part of a whole tissue

• they respond to signals from other cells and from the environment

• these signals are often chemical

why do cells communicate

• secreted signals can be local or long distance

• local signaling:

  • signals act on nearby target cells

    • growth factors such as fibroblast growth factor — FGF₁ (paracrine)

    • neurotransmitters such as acetylcholine - ACh (synaptic)

    • can act on the signalling cell (autocrine)

• long distance signaling:

  • signals act from a distance

    • hormones secreted from endocrine cells travel via circulatory system to act on target cells

    • e.g. insulin secreted from pancreatic beta cells enter bloodstream and travels and is detected by various body cells

outline the differences between local signaling and long distance signaling

1. reception

   • signalling protein (primary messenger) binds to a receptor protein

   • results in shape and/or chemical state change in the receptor protein

2. transduction

   • altered receptor activates a another protein, e.g. G-protein/adenylyl cyclase

   • the activated protein (often an enzyme) may cause a relay of changes

   • relay molecules known as ‘secondary messengers’, e.g. cAMP, IP₃

   • multiple other proteins may be activated

   • each activated protein causes a series of changes, this is often via phosphorylation — known as a phosphorylation cascade

3. response

   • all of the activated protens cause one or more functions to occur in the cell

   • this is where the cell actually does something

outline the three main steps of cell signalling

• the human body will simulataneously send out many different chemicals and molecules, all aimed at eliciting specific responses BUT only the target receptors will interact with that signal (ligand) and use it to activate signal transduction pathways

• specifity comes from the 3D molecular shape of the proteins involved — structure determines function

• exquisite control is possible: only certain cells at certain times will have particular receptors (i.e. dynamic), meaning that while the signal might be widespread the transmission of the signal occurs only where it is needed

describe the specifity of receptors

• receptors for water soluble molecules are membrane bound

  • e.g. G protein couped receptor, receptor tyrosine kinase, ligand-gated ion channel

• receptors for lipid soluble molecules are not membrane bound

  • can be located in the cytoplasm or inside the nucleus

  • e.g. lipid soluble hormones such as testerones, estrogen, progesterone, thyroid hormones bind to receptors within the cytoplasm and move to nucleus as a complex

outline where receptors are located

• transmembrane proteins — pass PM 7 times

• hundreds of different GPCRs exist

• many different ligands

• diverse functions:

  • e.g. development, sensory reception (vision, taste, smell)

• GPCRs couple with G proteins

  • G proteins are molecule switches which are either on or off depending on whether GDP or GTP is bound

  • (GTP: guanosine triphosphate, similar to ATP)

describe G-protein coupled receptors (GPCRs)

1. At rest, reeptor is unbound and G Protein is bound to GDP. The enzyme is in an inactive state

2. Ligand binds receptor, and binds the G protein. GTP displaces GDP. The enzyme is still inactive

3. Activated G Protein dissociates from receptor. Enzyme is activated to elicit a cellular response

4. G Protein has GTPase activity, promoting its release from enzyme, reverting back to resting state

describe the process of G-protein coupled receptors (GPCRs) being activated

• these channel receptors contain a ‘gate’

• binding of ligand (e.g. neurotransmitter) at specifc site on receptor elicits change in shape

• channel opens/closes as the receptor changes shape

• ions can pass through channel (e.g. Na+, K+, Ca2+, and/or Cl-)

• receptor — a molecule/protein which responds to a specific ligand

• ligand — a signalling molecule that binds specifically to another protein

• ion channel — memmbrane protein through which specific ions can travel

• ion channel receptor — membrane protein through which specific ions can travel, in response to ligand binding (also known as ionotropic receptors)

describe ligand gated ion channels/receptors

1. at rest, ligand is unbound and gate is closed

2. upon ligand binding, gate opens, specific ions can flow into cell

3. following ligand dissociation, gate closes, back to resting

• the nervous system heavily relies on ligand gated ion channels

  • the nervous system releases neurotransmitters and bind as ligands to ion channels on target cells to propagate action potentials

describe the process in which ligand gated ion channels/receptors work

• signals relayed from receptors to target molecules via a ‘cascade’ of molecular interactions

• protein kinases are enzymes that transfer a phosphate group from ATP to another (specific) protein (kinases phosphorylate), typically, this activates the protein

• series of protein kinases each adding a phosphate to the next kinase

• phosphates are enzymes that dephosphorylate (remove the phosphate) rendering the protein inactive, but recyclable

• typically, it is serine or threonine residues that are phosphorylated

• this means that mutations affecting these residues could be detrimental

describe transduction pathways

• sometimes another small molecule is included in the cascade, these are second messengers

  • e.g. cAMP and calcium ions

• recall earlier GPCR slide, plus:

  • the activated enzyme is adenylyl cyclase converts ATP to cAMP

  • cAMP acts as a secondary messenger and activates downstream proteins, for example, PKA which phosphorylates other proteins

outline cAMP as a second messenger

• low [Ca2+] inside cell (typically ~100nm)

• very high [Ca2+] outside the cell (more than 1000-fold higher)

• maintenance of concentration via calcium pumps is important

  • out of cell

  • into ER

  • into mitochondria

outline calcium as a secondary messenger

• here, the activated proteinis phospholipase C which then cleaves PIP2 (a phospholipid) into DAG and IP3

• IP3 diffuses through cytosol and binds to a gated channel in the ER

• calcium ions flow out of the ER down a concentration gradient and activate other proteins toward a cellular response

describe the role of Ca2+ and IP3 in GPCR signalling

• amplifies the response

• provides multiple control points

• allows for specificity of response

  • temporal

  • spatial

    • despite molecules in common

• allows for coordination with other signalling pathways

why are there so many steps to transduction

• examples of a cellular response include activation or regulation of:

  • gene expression

  • alteration of protein function to gain or lose an activity

  • opening or closing of an ion channel

  • alteration of cellular metabolism

  • regulation of cellular organelles or organisation

  • rearrangement/movement of cytoskeleton

  • a combination of any of these

• the transduction of a signal leads to the regulation of one or more cellular activities

what are examples of cellular responses

• all of the signals are for a limited time: activation usually promotes the start of deactivation, so that signalling is of short period of time, ensuring homeostatic equilibrium

• it means the cell is ready to respond again if required

• cAMP is broken down by phosphodiesterase (PDE)

• caffeine blocks the action of pDE

• inhibition of specific PDE’s can also be a therapeutic approach

  • e.g. viagra — inhibits a specific cGMP — degrading PDE

outline the importance of a response being turned off

• adrenalin acts through GPCR, activates cAMP and two protein kinases in a phosphorylation cascade

• results in active glycogen phosphorylase which can convert glycogen to glucose 1-phosphate

• amplification means that 1 adrenalin molecule can result in 10⁸ glucose 1-phosphate molecules

outline how adrenalin stimulates glycogen breakdown

• glycogen is a long term energy store in liver and skeletal muscle

• glycogen breakdown results in glucose 1-phosphate

• glucose 1-phosphate is then converted to glucose 6-phosphate which can then be used in glycolysis to generate ATP

outline how a large amount of ATP is generated quickly

• angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the coronavirus (SARS-CoV-2)

• surface spike glycoprotein (S protein)

• here, ACE2 in our respiratory tract is the lock, and the S-protein on the virus is the key

outline how receptors can be deceived

the process of going from DNA to a functional product (typically a protein)

what is gene expression

an organisms’s hereditary information

what is a genotype

actual observable or physiological traits

what is a phenotype

our genotype and its interaction with the environment

what determines our phenotype

• DNA (deoxyribonucleic acid) is the heritable material that is used to store and transmit information from generation to generation

• RNA (ribonucleic acid) acts as a messenger to allow the information stored in the DNA to be used to make proteins

• proteins carry out cellular functions

• three main steps:

  • transcription of RNA from DNA

  • processing of the pre-mRNA transcript

  • translation of the mRNA transcript to a protein

where does gene expression happen and what happens during the process of gene expression

three steps:

1. initiation: polymerase binds to promoter

2. elongation: moves downstream through the gene, transcribing RNA

3. termination:detaches after terminator reached

RNA uses the nitrogenous base Uracil, in place of Thymine and it is single stranded, while DNA is double stranded

outline transcription

• assembly of multiple proteins required before transcription can commence

• TATA box typically ~25nt upstream found in the promoter region

• assembly of several transcription factors including the TATA box binding protein (TBP) bind to DNA

• RNA Pol II can now bind along with more transcription factors to form the transcription initiation complex, and so transcription begins

• initiator tRNA = tRNA carrying methionine (Met)

• small ribosomal subunit with initiator tRNA already bound binds 5’ cap of mRNA

• small ribosome subunit scans downstream to find translation start site (AUG)

• hydrogen bonds form between initiator anticodon and mRNA

• large ribosomal subunit then binds — completing the initiation complex

• energy (GTP — guanosine triphosphate) is required for assembly

outline the initiation process of transcription

• 10-20 nucleotides exposed at a time when DNA unwound

• elongation: complementary RNA nucleotides added to 3’ end of growing transcript (3’OH of transcript binds with 5’ phosphate of incoming nucleotide) — It forms a phosphodiester bond

  • codon recognition:

    • base pairs with complementary anticodon GTP invested to increase accuracy/efficiency

  • peptide bond formation: '

    • a large subunit rRNA catalyses peptide bond formation

    • removes it from tRNA in P site

  • translocation:

    • moves tRNA from A to P site

    • tRNA in P site moves to E and is released

    • energy is required

  • empty tRNAs are ‘reloaded’ in the cytoplasm using aminoacyl-tRNA synthetases

• double helix reforms as transcript leaves the template strand

• termination: after transcription of the polyadenylation signal (AAUAAA) nuclear enzymes release the pre-mRNA and RNA polymerase then dissociates from the DNA

  • ribosome reaches a stop codon on mRNA

    • mRNA stop codon in the A site is bound by a release factor

  • release factor promotes hydrolysis

    • bond between p-site tRNA and last amino acid is hydrolysed, releasing polypeptide

  • ribosomal subunits and other components dissociate

    • hydrolysis of two GTP molecules required

    • ribosome components can be recycled

• fidelity (proofreading) is less than for DNA replication

• the pre-mRNA transcript is now ready for further processing

outline the elongation and termination process of transcription

• capping: a modified guanosine nucleotide is added to the 5’ end

• tailing: 50-250 adenosine nucleotides (polyA) are added to the 3’ end

• capping and tailing are thought to facilitate export, confer stability and facilitate ribosome binding in cytoplasm

• splicing: introns are removed from the transcript, typically making mRNA much smaller than Pre-mRNA

• definitions to know:

  • exons: regions that remain in mature RNA (includes UTR)

  • UTR: untranslated regions of 5’ and 3’ ends of mRNA

  • introns: intervening regions that do not remain in mature RNA

outline the second step of mRNA processing — capping, tailing, and splicing

• splicing occurs at the spliceosome, within the nucleus

  • spliceosome: a large complex of proteins and small RNAs

• introns are removed from the transcript and exons are rejoined to form mature mRNA

• alternative splicing is a process by which different combinations of exons are joined together, this results in the production of multiple forms of mRNA from the same pre-mRNA population

• alternative splicing allows for multiple gene products from the same gene

  • ~20,000 genes, there could be many times that number of proteins

where does splicing occur

• protein sequence determine its final structure

• structure determines function

• DNA mutations can affect ability of the protein to function

outline how protein sequence determines the function

• mature mRNA transcript exits nucleus and is bound by the ribosome

• codons are translated into amino acids

• tRNA molecules within the cytosol with specific anticodons carry corresponding amino acids

• hydrogen bonds form between mRNA and antidcodon of the appropriate tRNA

• the amino acid is added via peptide bonds to the growing polypeptide chain

outline translation

• tRNA and mRNA are held within a ribosome to enable the formation of the polypeptide

• mRNA binding site on small subunit

• A site: holds ‘next in line’ tRNA

• P site: holds tRNA carrying the growing polypeptide

• E site: tRNAs exit from here

outline the ribosome binding sites for mRNA and tRNA

tRNA is the physical link between the mRNA and the amino acid sequence of proteins

what is the role of tRNA

• multiple control points:

  • transcription factors need to assemble, and DNA needs to be accessible

  • capping, extent of polyadenylation, alternate splicing, producing an mRNA able to be translated

  • specific proteins assist in nuclear export of mRNA

  • regulatory proteins can block translation, variable mRNA life-spans

outline why gene expression is tightly regulated

• to achieve the right thing at the right time in the right place (this is temporal and spatial control)

• housekeeping (commonly used) proteins are continuously produced

  • protein and mRNA are present in large quantities (e.g. Tubulin)

  • typically, have longer ‘half-life’ in cells

• other proteins are produced in response to stimuli as required

  • cell signaling (e.g. ligand binding to cell surface receptor, or activiating an intracellular receptor)

  • signal transduced and may enter nucleus to activate transcription

  • results in the production of a short-lived protein to carry out the required function

why is control of gene expression important

• the side chains (R groups) determine the properties of each amino acid

• they collectively determine the final structure and function of the protein

• there are twenty standard (coded for) amino acids

amino acid properties

• protein sequence (primary structure) is determined by DNA sequence

• peptide bonds are covalent bonds between amino acids (relatively strong)

• the polypeptide starts to form secondary structures as soon as it leaves the ribosome

describe the primary structure of amino acids

• secondary structure:

  • held by weak hydrogen bonds to form alpha helix and beta sheets

• tertiary structure:

  • 3D shape stabilised by side chain interactoins

• quaternary structure:

  • multiple proteins associate together to form a functional protein

  • not all proteins form quaternary structures

describe the secondary, tertiary, and quaternary structures of amino acids

• all translation commences on free ribosomes

• many proteins are processed and sorted through the RER and Golgi — but not all

• proteins destined to function in the cytosol — complete translation on free ribosomes

• proteins that go through the endomembrane system — complete translation at fixed ribosomes on the RER

outline protein processing and sorting

• signal peptide:

  • at N terminus of the protein (~20aa)

• SRP: signal recognition particle

  1. polypeptide synthesis begins

  2. SRP binds to signal peptide

  3. SRP binds to receptor protein

  4. SRP detaches and plypeptide synthesis resumes

  5. signal-cleaving enzyme cuts off signal peptide

  6. completed polypeptide folds into final conformation

• at step 6:

  • a secretory protein such as insulin is solubilised in lumen, while a membrane protein remains anchored to the membrane

  • both then go to the Golgi via vesicles for further maturation

describe how signal peptides direct ribosomes to RER

• translation is now complete, but the protein may not yet be functional

• common (there are 100s) post translational modifications include:

  • phosphorylation (addition of a phosphate group)

  • methylation (addition of a methyl group)

  • acetylation (addition of an acetyl group)

  • biotinyation (addition of biotin)

  • carboxylation (addition of a carboxylic acid group)

  • carbohydrate addition (particulary for membrane bound proteins, e.g. glycoproteins)

  • cleavage

  • ubiquitination

• some occur within the Golgi, others in the cytosol

• can confer activity — e.g. via phosphorylation or enzyme cleavage

  or ability to interact with other molecules — e.g. biotinylation, methylation of histones

  or direct to particular locations — e.g. ubiquitination for proteasome degradation

outline post-translational modifications to proteins

• human cells are diverse and have different destinies

• a cell has three possible destinies:

  • live and function without dividing

  • grow and divide

  • die

• various signals tell a cell which path to take

cell diversity and cell destiny

• somatic cell division: mitosis — diploid (2n) to diploid (2n)

• reproductive cell division: meiosis — diploid (2n) to haploid (1n)

what are the two different types of cell division

• G1: growth or gap phsae 1

  • most cellular activities are occurring here

  • duration variable — cell type specific

• S: synthesis of DNA

  • DNA replication occurs strands are separated at the hydrogen bonds holding the nucleotides together new strand of DNA is synthesised opposite each of the old strands

• G2: growth or gap phase 2

  • checks for correct DNA synthesis prepares for the mitotic phase (synthesis of the proteins and enzymes required, gathering of reactants)

  • replication of centrosomes is completed

outline the interphase of the eukaryotic cell cycle

• mitotic phase = mitosis plus cytokinesis

• prophase:

  • mitotic spindle forms

  • two sister chromatids join together at the centromere to form the chromosome

  • fragments of nuclear envelope, condensed chromosome, and spindle tracks visible

• metaphase:

  • condensed chromosomes aligned

• anaphase:

  • separated chromosomes

• telphase and cytokinesis:

  • nuclear envelope forming

  • cleavage furrow

describe the mitotic phase

• during interphase, DNA replicates

• during prophase, DNA condenses

  • two identical chromatids per chromosome

  • these are called sister chromatids

• during metaphase, chromosomes ‘line’ up

• during anaphase, sister chromatids separate before the nuclear envelope refors in telphase

• daughter cells are ‘identical’ to parent cell

• human diploid cells have 46 chromosomes, 23 from each parent

what is a sister chromatid

• G1 checkpoints:

  • is the DNA undamaged?

  • is cell size and nutrition ok?

  • appropriate signals present?

    • if not — may exit to G0

• M checkpoints:

  • are all chromosomes attached to spindles

multiple signals required to pass G1 and M checkpoints

• occurs in the gonads (ovaries and testes)

• produces gametes which are haploid (a single set of 23 chromosomes)

• fertilisation then restores the diploid number of chromosomes (2n)

• produces cells genetically different from the parent cell

• there are two stages of meiosis:

  • meosis I:

    • prophase I (synapsis and crossing over, tetrads form)

    • metaphase I (pairs of homologous chromosomes)

    • anaphase I (sister chromatids remain attached)

    • telophase I

  • meiosis II:

    • prophase II

    • metaphase II

    • anaphase II

    • telophase II

outline meiosis

• meiosis I separates homologous chromosomes

• synapsis: two sister chromatids of each pair of homologous chromosomes pair up

• the 4 chromatids are called a tetrad

• non-sister chromatids within these tetrads may then cross over: causes recombination

• prophase I:

  • sister chromatids present

  • centrosome (with centriole pair) present

  • crossing over occurs at the chiasmata

  • spindle micro-tubules present

  • fragments of nuclear envelope visible

  • pairs of homologous chromosomes present

• metaphase I:

  • kinetochore (at centromere)

  • kinetochore microtubules

  • metaphase plate

• anaphase I:

  • sister chromatids remain attached

  • homologous chromosomes separate

• telophase I and cytokinesis:

  • cleavage furrow

meiosis I — separates homologous chromosomes

• very similar to mitosis, except not preceeded by DNA replication

• sister chromatids separate in anaphase II

• haploid daughter cells form in telphase II and cytokinesis

meiosis II — separates sister chromatids

• mitosis:

  • prophase:

    • chromosome duplicated

  • metaphase:

    • individual chromosomes line up

  • anaphase/telophase:

    • sister chromatids separate

• meiosis

  • prophase I:

    • crossing over at chiasma

    • chromosome duplication results in pair of duplicated homologs

  • metaphase I:

    • pairs of homologous chromosomes line up

  • anaphase I/telophase I:

    • homologs separate

    • sister chromatids separate

outline the differences between mitosis and meiosis during each of their processes

• mitosis

  • DNA replication: occurs during interphase before mitosis begins

  • number of divisions: one, including prophase, prometaphase, metaphase, anaphase, and telophase

  • synapsis of homologous chromosomes: does not occur

  • number of daughter cells and genetic composition: two, each genetically identical to the parent cell, with the same number of chromosomes

• meiosis:

  • DNA replication: occurs during interphase before meiosis I but not meiosis II

  • number of divisions: two, each including prophase, metaphase, anaphase, and telophase

  • synapsis of homologous chromosomes: occurs during prophase I along with crossing over between nonsister chromatids; resulting chiasmata hold pairs together due to sister chromatid cohesion

  • number of daughter cells and genetic composition: four, each haploid (n); genetically different from the parent cell and from each other

compare the properties of mitosis to meiosis

• sources of genetic variation:

  • independent assortment at metaphase I (2³³ > 8 million possible combinations)

  • crossing over at prophase I (~1-3 crossover events per pair)

  • fusion between two gametes (> 2³³ times 2³³ combinations)

where do sources of variation occur from

• mutations can affect the structure and function of a protein

• altered DNA sequence can have major effects on resulting protein function

  • germ line — passed on to future progeny

  • local/somatic — during cell division, not whole body — local effects

• large scale alterations — chromosomal rearrangements

• small scale alterations — one or a few nucleotides altered

• small scale mutations can be:

  • substitutions — where one base is replaced by another - can have minimal or major effect

  • insertions/deletions — can have major effect if within coding sequence - can cause a frameshift

what is the effect of DNA sequence changes

• substitutions can be:

  • silent

  • missense

  • nonsense

• insertions or deletions (indels):

  • cause frameshift if 1 or 2 nt

  • can maintain frame if 3 nt

outline substitutions and indels in protein coding regions

GGC codon becomes GGU codon, but still codes for Glycine — so no effect on protein

name an example of a silent mutation

• GGC codon becomes AGC codon, so Gly becomes Ser — effect depends on residue role

• Gly becomes Ser

  • hydrophobic becomes hydrophilic

  • it could impact ability to remain embedded in a membrane

what is an example of a missense mutation

• AAG codon becomes UAG codon, so Lys becomes a STOP — truncated protein

what is an example of a nonsense mutation

AAG codon becomes UAA codon, so Lys becomes STOP — truncated protein

what is an example of a frameshift mutation via insertion

• UUU codon becomes UUG, so Phe becomes Leu, plus downstream residues

  • protein is completely altered from point of frameshift, can have catastrophic effect

what is an example of a frameshift mutation via deletion

AAG codon is lost (Lys), but downstream residues are intact — frame is maintained

what is an example of a 3 nucleotide-pair mutation