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Protein Traffic and Signal Recognition in Cellular Compartments, Summaries of Biology

An overview of protein traffic between different cellular compartments, focusing on the recognition of signal sequences by srp and the transport of proteins through nuclear pores and mitochondrial membranes. It also covers the role of snares and rabs in vesicular transport and the regulation of coat assembly and vesicle stability.

Typology: Summaries

2023/2024

Available from 04/08/2024

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lOMoARcPSD|39591929
G. J. Tortora, B. H. Derrickson,
Biology and physiology
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Download Protein Traffic and Signal Recognition in Cellular Compartments and more Summaries Biology in PDF only on Docsity!

lOMoARcPSD|

G. J. Tortora, B. H. Derrickson,

Biology and physiology

Table of Contents Lecture 15 Intracellular Transport ................................................................................................................. 2 Covered in presentation but not in lecture ............................................................................................... 3 Lecture 16 ...................................................................................................................................................... 4 Lecture 17 ...................................................................................................................................................... 7 Lecture 18 .................................................................................................................................................... 13 Key: Text Topics/details covered in lecture but not seemingly relevant to exam material, thus skipped Text Topics/details not necessary to know for exam

▪ A cytosolic protein , RAN-GAP (RAN GTP-ase activating protein) activates hydrolysis of GTP attached to RAN. ▪ This creates a gradient of RAN-GTP across the nuclear pore – with more RAN GTP inside the nucleus than outside. ▪ RAN-GTP binds to nuclear import receptors after they diffuse through the nuclear pore and into the nucleus ▪ causes them to release their cargo proteins, which therefore accumulate inside the nucleus. ▪ RAN-GTP has the opposite effect on nuclear export receptors , causing them to bind their cargo. ▪ They then diffuse through the pore into the cytosol. ▪ Import depends on the receptor NOT binding to RAN when it binds to its cargo. When the receptor gets into the nucleus it binds to the RAN and release cargo ▪ Export relies on both receptor and RAN bound at the same time in the nucleus, both are released when receptor gets into cytosol ▪ Focus on nuclear import o Nuclear transport as a means of gene regulation ▪ The activity of some gene regulatory proteins is controlled by keeping them out of the nuclear compartment until they are needed there. ▪ In many cases, this control depends on the regulation of nuclear localization and export signals ; these can be turned on or off, often by phosphorylation of adjacent amino acids. o Ex. NFAT and Nuclear Transport ▪ T cell activated via antigen binding ▪ Ca2+^ levels increase due to Ca2+^ channel opening ▪ This rise in Ca2+^ levels activates the protein phosphatase, calcineurin ▪ Calcineurin dephosphorylates NF-AT ▪ Dephosphorylation of NFAT causes a conformational change which exposes a nuclear import sequence on the protein’s surface. ▪ NFAT enters the nucleus, where it triggers gene expression appropriate to the T- cells role in the immune response. Covered in presentation but not in lecture

  • Movement of proteins between cellular compartments: Transmembrane transport o protein traffic between the cytosol and an organelle that is topologically different ▪ Cytosol → ER ▪ Cytosol → mitochondria - Neither the mitochondrial nor chloroplast genomes contain information necessary to code for all of their proteins. - They rely on the import of their proteins from the cytosol following synthesis. o occurs through membrane-bound protein translocators ▪ The transported protein molecule usually must unfold to snake through the translocator
  • Movement of proteins between cellular compartments: vesicular transport

o protein traffic among topologically equivalent organelles ▪ ER ↔ Golgi ▪ Golgi ↔ Endosomes ▪ Endosomes ↔ Lysosomes ▪ Endosomes ↔ Plasma Membrane o occurs through membrane-enclosed transport intermediates called VESICLES

  • Newly synthesized (precursor) Mitochondrial Proteins in the cytosol are surrounded by protein- folding chaperones that prevent them from aggregating. o Most common chaperone: Hsp
  • Mitochondrial versions of these chaperones also exist and help these precursor proteins fold into 3D structures once they enter the mitochondria. Lecture 16 10/25/
  • Mitochondrial transport o Mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria o Most of the mitochondrial precursor proteins have a signal sequence at their N terminus that, when folded forms an amphipathic alpha helix o Charged residues cluster on one side o Uncharged residues cluster on the other side o Protein translation across mitochondrial membranes is mediated by multi-subunit complexes that function as protein translocators: ▪ The TOM complex functions across the outer membrane ▪ Two TIM complexes, the TIM23 and TIM22 complexes, function across the inner membrane
  • Mitochondrial protein translocators o ALL nucleus-encoded mitochondrial proteins must first enter via TOM o Helps insert transmembrane proteins into outer mitochondrial membrane o Transmembrane proteins with a β-barrel structure are transferred to the SAM complex for proper folding. o TIM 23 spans both outer and inner mitochondrial membranes o Transports: ▪ soluble proteins into MATRIX ▪ membrane proteins into inner mitochondrial membrane o Import ATPase complex binds to and pulls proteins through TIM23 channel
  • Proteins entering the ER undergo CO-translational translocation o imported into the ER as they are being synthesized ▪ no requirement for additional ATP to pull the protein into the ER ▪ as you extend the protein, the force generated by extending the protein pushes it across the transporter into the ER o What types of proteins require co-translational translocation? ▪ Water soluble (non membranous) proteins destined to:
  • Coat Proteins Represent the Initial Step in Vesicle Formation o Name a protein coat complex – Clathrin, COPI, COPII o Transport vesicles bud off as coated vesicles that have a distinctive cage of proteins covering their cytosolic surface. o Before the vesicle fuses with a target membrane, the coat is discarded , to allow the two cytosolic membrane surfaces to interact directly and fuse. o Different coat proteins are involved in transport between different organelles: ▪ COPII coats ER-to-Golgi vesicles ▪ COPI coats vesicles moving from Golgi to ER, Golgi to plasma membrane (secretory vesicles), and within the Golgi ▪ Clathrin is mainly involved in transport to/from and within the endosomal compartments
  • Phospholipids containing inositol head groups mark organelles and membrane domains o Inositols can get phosphorylated at various locations by different lipid kinases (often located in distinct organelles, providing specificity)
  • (Phosphatidyl inositol) PI’s can recruit various proteins that possess lipid binding domains o These lipid binding domains usually only recognize a specific type of PI
  • Regulation of coat assembly and vesicle stability o Adapter proteins bind to membrane proteins and recruit coat proteins ▪ Often bind to cargo receptors – transmembrane proteins that bind soluble cargo proteins for transport o Coat recruitment GTPases control coat assembly ▪ Monomeric GTPases regulate many steps in vesicular traffic ▪ Sar- 1 regulates COPII assembly ▪ Arf proteins regulate COPI and clathrin assembly o Active Sar1-GTP then promotes the assembly of coat complexes. o GTP hydrolysis causes coat disassembly after budding. ▪ Sar1-GDP = cytosol = inactive ▪ Sar1-GTP = ER membrane-bound = active
  • Vesicle docking and targeting o Membrane traffic needs to proceed in an orderly way ▪ Transport vesicles must be highly selective in recognizing the correct target membrane with which to fuse. o Specificity in targeting: surface markers that identify vesicles according to their origin and type of cargo. o Target membranes display complementary receptors that recognize the appropriate markers.
  • Recognition of donor vesicles by acceptor membranes is controlled mainly by two classes of proteins: SNAREs and Rabs. o SNARE proteins ▪ provide specificity ▪ catalyze the vesicular fusion with the target membrane. ▪ v-SNAREs: vesicle ▪ t-SNAREs: target membrane

o Rabs (GTPases): ▪ Work together with other proteins to regulate the initial docking and tethering of the vesicle to the target membrane ▪ See Table 13- 1 for list of some Rabs

  • Entry into vesicles leaving the ER is usually a selective process o Cargo RecruitmentMembrane proteins have exit signals in their cytosolic ‘tails’ that are recognized by coat proteins ▪ Soluble Proteins bind to cargo receptors that have exit signals in their cytosolic ‘tails’
  • Transport from the ER to the Golgi Apparatus Is Mediated by Vesicular Tubular Clusters o Transport vesicles leaving the ER fuse together to form intermediate compartments called vesicular tubular clusters o These clusters ▪ travel towards the cis Golgi via motor proteins on microtubule tracks ▪ generate coated vesicles going back to the ER (COPI coat) – retrograde transport
  • ER retrieval signals: o Membrane ER resident proteins retrieval signals in their cytosolic tails ▪ recognized by COPI coat proteins o Soluble ER resident proteins ▪ Retrieval signals within their structure ▪ Bind to receptors ▪ EX: KDEL sequences bind to KDEL receptors
  • Two current models for how cargo travels through the Golgi: o CISTERNAE = STATIC ▪ Vesicles travel between them o CISTERNAE = DYNAMIC ▪ Move upward, changing their properties slightly as they migrate ▪ Vesicular tubular cluster becomes new cis face of golgi, old cis face becomes medial cisternae, old trans face becomes trans golgi network and can move away/bud off vesicles and turn over to become a new trans face Lecture 17 10/30/
  • Transport from the Trans Golgi Network to the Cell Exterior: Exocytosis o Many proteins leaving the Golgi need to get to the plasma membrane. o Vesicles carrying such proteins fuse with the plasma membrane via exocytosis. o Vesicle cargo ▪ membrane proteins and the lipids become part of the plasma membrane ▪ soluble proteins are secreted into the extracellular space.
  • Transport from the Trans Golgi Network to the Cell Exterior: Two Pathways o Constitutive secretory pathway ▪ Never-ending, occurring all the time o Regulated secretory pathway ▪ Proteins are sorted in the trans golgi network and retained for future use
  • The budding and internalization of vesicles from the plasma membrane is called endocytosis. o Vesicle cargo ▪ membrane proteins and the lipids are removed from the plasma membrane - some will be Recycled back to the surface - some will be Degraded ▪ soluble proteins - from extracellular space - carried in the lumen ▪ common membrane protein cargo: receptors (and their ligands) ▪ budding in from the plasma membrane is topologically equivalent to budding out from the ER/golgi
  • Types of Endocytosis o Distinguished on the basis of the size of the endocytic vesicles formed. ▪ Phagocytosis (“cellular eating”) - the ingestion of large particles , such as microorganisms or dead cells via vesicles called phagosomes (generally >250 nm in diameter). ▪ Pinocytosis (“cellular drinking”) - the ingestion of fluid and solutes - Vesicles called pinocytic vesicles (about 100 nm in diameter). - Includes receptor-mediated endocytosis o Most eukaryotic cells are continually ingesting fluid and solutes by pinocytosis
  • Ex. Cholesterol gets into cells via receptor-mediated endocytosis
  • Ex. Epidermal growth factor receptor (EGFR) gets degraded, along with its ligand, but transferrin receptor (TfnR) gets recycled to plasma membrane
  • Transcytosis: Molecules internalized at one end of a ‘polarized’ cell are transported to a different end.
  • Membranes as Barriers o Biological membranes serve as semipermeable barriers o Nonpolar molecules can diffuse freely, but polar/charged solutes are unable to cross without assistance o Transmembrane protein channels and transporters serve to allow polar/charged solutes to cross membranes ▪ Channels allow diffusion down a concentration gradient ▪ Transporters use conformational changes to move substrates across the membrane, and may transport down ( passive transport) or up ( active transport) a concentration gradient
  • Diffusion o HIGH concentration → LOW concentration ▪ Going from a more to less ordered state → increase in entropy o DOWN concentration gradient: ▪ concentration gradient– difference in concentration of a solute in one region compared to another o The process or movement of any molecule or ion moving down or up a concentration gradient requires a change in free energy
  • Which is true about diffusion? Molecules sometimes move towards other molecules and sometimes away from other molecules o Random motion will result in spreading out of molecules over time
  • Diffusion of CHARGED substances across cell membranes o Diffusion is net movement of charged solute down its electrochemical gradient o sum of: ▪ concentration gradient ▪ electrical gradient – electrical potential difference between 2 sides of membrane
  • Electrochemical differences are additive
  • So, ΔG (Gibbs free energy) can tell us whether a substance will move passively (no energy needed) or actively (energy input required) across a membrane. o If positive, the value of DG^ (net flux INTO cell) is the^ amount of energy required to move^ a mole of solute up its electrochemical gradient and into the cell. o If negative, DG^ (net flux INTO cell) is the maximum^ amount of energy available to drive another process that is coupled to the diffusion of a solute down its electrochemical gradient and into the cell.
  • Ways molecules can get across a membrane: simple diffusion o Diffusion across lipid bilayer (independent of proteins) o Limited by: ▪ PolarityChargeSize
  • Diffusion across membranes: Permeability o In the absence of proteins, the lipid bilayer allows free diffusion of select types substances down their concentration gradients: ▪ hydrophobic molecules ▪ Small polar molecules o Restricts the diffusion of other types of substances: ▪ ions ▪ larger polar molecules o The membrane = semipermeable barrier. o The membrane must be able to maintain concentration differences between the internal and external environments. o Health of the cell requires that material transport be a regulated process.
  • Transport Proteins o Cells have evolved membrane proteins whose function is to transport small molecules and ions. o Membrane proteins mediate 2 basic types of movement across cell membranes: ▪ Passive TransportActive Transport
  • Simple Diffusion vs Transporter-mediated diffusion o Simple diffusion:

▪ Temperature

  • temperature - sensitive channels
  • Passive transport – facilitated diffusion o Transport of neutral, polar molecules larger than water or urea, such as glucose - or charged molecules such as amino acids (rather than simple ions) o not coupled to any external energy source, such as ATP ▪ this is still passive transport even though it requires a shape change in the transporter to get across the membrane) ▪ the direction of net flux follows the electrochemical gradient for whatever molecule is transported. o solute molecule binds tightly to a highly specific site on the protein and causes a conformational change in the transporter protein. ▪ Transition between A and B is random/reversible, does not depend on whether solute binding sites are occupied. o Facilitated transport proteins: ▪ Like enzymes, do not alter DG for transport – movement is always down the electrochemical gradient for the solute - they just speed up movement ▪ Have a relatively slow turnover compared to channels
  • maximal transport rate ~1000 molecules per transport protein per second
  • compared to 1 million molecules for channels
  • Facilitated diffusion – Glucose transport o Humans have 5 related glucose facilitated transport proteins, One of these, GLUT4 is common to insulin-responsive cells o Insulin promotes the insertion of GLUT4 transporters into the membrane of target cells, promoting glucose uptake
  • Active transport o Movement of solutes UP their electrochemical gradients. o Can create and maintain concentration gradients of solutes across membranes. o Can sometimes directly contribute to membrane potentials (can be “electrogenic”) if the transported solutes carry a net charge across the membrane.
  • Types of active transport o Three main types, depending on the energy source ▪ ATP-ase pumps.
  • Couple movement of solutes to ATP hydrolysis ▪ Other pumps that use diverse energy sources (e.g. light, oxidation of NADH)Coupled transporters
  • link the movement of one solute up its electrochemical gradient to the movement of another solute down its electrochemical gradient. Their energy source is therefore an existing electrochemical gradient.
  • Coupled transporters o (Uniport: Transport one molecule) o Symport: Transport two molecules in the same direction o Antiport: Transport two molecules in different directions

Lecture 18 11/01/

  • Types of ATP-driven pump proteins: P-type Pumps o “P” stands for the use of a high energy phosphoprotein intermediate o Multipass transmembrane proteins o Pump H+, K+, Na+, Ca2+
  • P-type Pump Example 1: Ca2+ ATPase o Like Na+, Ca2+^ ions are kept at very low concentrations in the cell relative to the extracellular environment: ▪ Inside the cell: [Ca2+] ~ 0.1μM ▪ Outside the cell: [Ca2+] ~ 103 μM o Even the smallest Ca2+^ cytosolic influx can lead to a massive increase in cell levels. o Regulated Ca2+^ influxes form the basis of a variety of cell signaling events. o Helps maintain a very low resting Ca2+^ concentration in the cytosol. o Transports one Ca2+^ ion out of the cell for every ATP molecule consumed. o Plasma membrane, smooth ER, sarcoplasmic reticulum (muscle ER) o Creates a calcium ion store with in the smooth ER
  • P-type Pump Example 2: Na+/K+ Pump o Each cycle of the sodium/potassium pump (Na/K ATP-ase) transports: ▪ 3 Na+ out of a cell ▪ 2 K+ into a cell - Causes a net +1 charge out of the cell per cycle - Electrogenic – producing a change in the electrical potential ▪ All for the hydrolysis of only one ATP molecule o The pump has specific binding sites for sodium and potassium. o The binding and release of sodium and potassium ions on either side of the membrane is coupled to ATP-hydrolysis o Each step in the hydrolysis of ATP and the binding of the transported ions drives conformational changes that create a pumping cycle by alternately exposing the ion binding sites to one side of the membrane and then the other. o Na+ binds o ATP hydrolysis leads to phosphorylation of the pump o Conformational change → release of Na+ o Binds K+ o Causes dephosphorylation of the pump o Conformational change o Unphosphorylated pump has higher affinity for Na+
  • Types of ATP-driven pump proteins: V-type Pumps o Made up of multiple subunits o Use ATP, but not via a phosphorylated intermediate o (the phosphate is released, not added to the pump). o V type pumps are found in membranes of: ▪ Lysosomes ▪ Synaptic vesicles
  • T-helper cell & B cell
  • Signaling molecules: Overall action o #1: Extracellular signals can act slowly or rapidly to alter the function of a target cell o #2: Each cell is programmed to respond to specific combinations of extracellular signal molecules - live or die ▪ Most of the cells in a complex animal are also programmed to depend on a specific combination of signals simply to survive. ▪ When deprived of these signals (in a culture dish, for example), a cell activates a suicide program → apoptosis ▪ Because different types of cells require different combinations of survival signals, each cell type is restricted to different environments in the body. o #2: Each cell is programmed to respond to specific combinations of extracellular signal molecules - divide, differentiate, do ▪ A typical cell in a multicellular organism is exposed to hundreds of different signals in its environment. ▪ These signals can act in many millions of combinations. ▪ A cell may be programmed to respond to one combination of signals by growth/division ▪ to another combination by differentiating ▪ and to yet another by performing some specialized function such as contraction or secretion. o #3: Different cells can respond differently to the same extracellular signal molecule – cells interpret signals ▪ Cellular responses vary according to:
  • Unique collection of receptor proteins the cell possesses
  • the intracellular signaling machinery by which the cell integrates and interprets what it receives ▪ determine the particular subset of signals to which a cell can respond ▪ The same signal molecule often has different effects on different target cells. ▪ Ex. acetylcholine o #4: The same signaling molecule can have different concentration-dependent effects on the same cell type ▪ So the AMOUNT of signal can dictate what happens within the same cell. ▪ Embryonic Development
  • molecules called morphogens diffuse out from signaling centers in developing tissues, creating a morphogen concentration gradient.
  • Cells adopt different fates depending on their position in the gradient. In this way layers of cells develop, each with a different function
  • Cell communication is like human communication. Paracrine signaling is like talking to people at a cocktail party
  • Signaling molecules: types of signals o Cells are specialized to receive and respond to a wide variety of stimuli o Mechanical ▪ adhesion to substrates, membrane distortion, sound

o Light o Heat o Chemical ▪ amino acids, small peptides, and proteins ▪ nucleotides ▪ steroids ▪ fatty acid derivatives ▪ dissolved gases: nitric oxide, carbon monoxide.

  • Lipid soluble signals o Many signaling molecules are lipid soluble and can simply diffuse across the plasma membrane. o Example: steroid hormones & gaseous signaling molecules (e.g. nitric oxide) o Steroid hormones vary in chemical structure, but all are synthesized from cholesterol. o Cortisol – adrenal glands ▪ Influences metabolism o Sex hormones – ovaries and testes ▪ Determine secondary sex characteristics that distinguish males and females o Vitamin D – skin in response to sunlight ▪ Regulates Ca2+^ uptake/excretion o Thyroxine (non-steroid, tyrosine derivative)– thyroid glands ▪ Regulates metabolism
  • Diffusion across membranes: Permeability o In the absence of proteins, the lipid bilayer allows free diffusion of select types substances down their concentration gradients: ▪ hydrophobic molecules ▪ Small polar molecules o Restricts the diffusion of other types of substances: ▪ ions ▪ larger polar molecules o Lipid soluble signals and their receptors ▪ Steroid hormones: - travel to their target cells via carrier proteins - bind to intracellular receptors, which can either be cytosolic or nuclear
  • Intracellular receptors that bind hormones o The receptors for steroid hormones are members of the nuclear receptor subfamily o Prior to ligand binding, these receptors are bound to an inhibitory protein and are inactive o Ligand binding causes a conformational change in the receptor, which causes the inhibitory protein to dissociate. o Exposes a site which binds to the promoter region upstream of a specific targeted gene. o Transcription of that gene is then increased, producing specific proteins that cause changes in cell behavior o Different cells respond differently to hormones because: ▪ Only certain cells have nuclear receptors for each hormone

o Enzymes activated by a receptor protein often form protein complexes called ‘signaling complexes’, which regulate Speed/Efficiency/specificity of a cellular response o These complexes can be organized around a “scaffold” protein o These complexes can be assembled following receptor activation o These complexes can be assembled on phosphorylated phosphoinositide lipids

  • The nature of signaling pathways: Intracellular signaling molecules : signaling complexes o These complexes can be ▪ Organized around a “scaffold” protein ▪ Assembled following receptor activation ▪ Assembled on phosphorylated phosphoinositide lipids
  • Receptors and their response to signals o Enzyme-coupled receptors: Receptor Tyrosine Kinases ▪ Activation of RECEPTOR TYROSINE KINASES occurs after ligand binding through dimerization and cross-phosphorylation
  • Signaling through Receptor Tyrosine Kinase o Proteins with SH2 domains then bind the phosphotyrosine residues and lead to activation of signaling proteins. o One of these signaling proteins can be Phospholipase C ▪ Ca2+ release likely also mediates other effects not shown here! ▪ Note that this downstream process can be linked to some other upstream event (doesn’t HAVE to be RTK)
  • Signaling through G-protein Coupled Receptors o GPCRs are the most common drug targets o G = Guanosine-5'-triphosphate (GTP) o GPCRs are bound to heterotrimeric G-proteins o Without Ligand = Inactive GPCR & G protein bound to GDP o With Ligand = Active GPCR & G protein bound to GTP o Upon activation, bg subunits are separated from the a subunit
  • The Role of Calcium as an Intracellular Messenger o Ca2+^ ions have a key role in a remarkable variety of cell activities: ▪ muscle contraction, ▪ cell division, ▪ secretion (exocytosis), ▪ endocytosis, ▪ fertilization, ▪ synaptic transmission, ▪ metabolism, ▪ cell movement
  • Calcium ion concentration in the cytosol increases upon activation of ligand-gated Ca2+ ion channels in the ER by the second messenger inositol triphosphate. Lecture 19 11/06/
  • Replication is semi-conservative o Each DNA strand is used as a template for the synthesis of a complementary strand

o Damaged DNA, if unrepaired, can persist through cell divisions. o Errors in copying, if unrepaired, are propagated through cell divisions

  • Replication occurs in the 5’ to 3’ direction o New nucleotides are added at the 3’ end. o Chain growth occurs in the 5’ to 3’ direction. o Addition of deoxynucleotides requires a primer. ▪ (telomeres are the exception) o This mode of replication results in the antiparallel double helix structure. o DNA replication is catalyzed by DNA polymerase – an enzyme with fingers , palm and thumb domains. o 5’ to 3’ polymerization occurs with an error rate of 1 in 105 nucleotides. Other processes reduce the error rate further.
  • Solutions for the 1 st^ three problems of replication o (1) Strand polarity ▪ All synthesis is 5’ to 3’. Okazaki fragments are synthesized on the lagging strand. DNA Ligase seals gap between successive fragments. o (2) Unzip DNA ▪ A hexameric complex called DNA helicase unzips the DNA. It uses ATP and acts as a rotary engine. Unzipped DNA is stabilized by single-stranded DNA binding protein. o (3) Processivity ▪ A sliding clamp holds the DNA polymerase in place. The clamp is loaded on DNA by a clamp loader that uses ATP hydrolysis to lock the clamp around DNA.
  • At the replication fork, the polymerases on both the leading strand and the lagging strand are moving in the same direction.
  • Rapid rotation of DNA is needed ahead of the replication fork o Problem (4): How to untangle the DNA? o Solution: Topoisomerases relieve torsional stress ▪ Changes the topology of DNA
  • Topoisomerase I: nick and swivel mechanism o Topoisomerase I does not require ATP to relieve strain
  • Topoisomerase II: gating mechanism o Topoisomerase II requires ATP to untangle DNA. o The enzyme attaches covalently to both strands of one DNA helix, creating a gap in the DNA helix. o The other DNA helix is then passed through the gap.
  • What do the enzymes topoisomerase I and topoisomerase II have in common? They both have nuclease activity
  • DNA replication begins at replication origins o A region of high AT content
  • Problem (5): One copy – Prokaryotic solution o Prokaryotic origins have a refractory period o Methylation is mediated by the Dam methylase in prokaryotes
  • What about in eukaryotes? It’s more complex…