Note
Studied by 2 peopleCells - CTR - Week 1
Topic 1: CELL STRUCTURE & FUNCTION 1 CTR The Work of David S. Goodsell Structural Biologist & Artist Scripps Research Institute, La Jolla, California This image is a panoramic view of the interior of a eukaryotic cell, such as a cell from your own body. The area covered is shown in the schematic map to the right. The panorama starts at the cell surface, passes through an area of cytoplasm, then follows the synthesis of proteins from the endoplasmic reticulum, through the Golgi, and into a coated vesicle. At the center of the panorama is a mitochondrion, generating energy for the cell. The final region passes into nucleus. All macromolecules are shown, with proteins in blue, DNA and RNA in red and orange, lipids in yellow, and carbohydrates in green. Ribosomes, composed of RNA and protein, are colored magenta. In a real cell, the spaces between each macromolecule are filled with small molecules, ions and water. Published in Moran, L.A. and Scrimgeour K.G. (1994) "Biochemistry" Neil Patterson Publishers /Prentis Hall, North Carolina. 2 © 1994 Neil Patterson Publishers Topic 1 Learning Outcomes ▪ In the notes – the first section is Learning Outcomes ▪ But you may see them referred to Learning Objectives at the start of the week in lectures. ▪ When studying always tick off each individual learning outcome once you can successfully do it. ▪ The exam questions are designed to test the learning outcomes. 3 Example 2) Describe the structure and function of the plasma membrane and explain the various forms of cellular transport Example Exam Question: Name two molecule types that are embedded in the plasma membrane which protrude into the extracellular space (2 marks) 4 Topic 1 Learning Objectives By the end of this Topic 1, students will be able to: 1. Describe the major differences between prokaryotes and eukaryotes 2. Describe the structure and function of the plasma membrane and explain the various forms of cellular transport 3. State the components of the cytoplasm and describe the structure and function of the cytoskeleton, centrioles, cilia and flagella 4. Discuss the structures and functions of the following organelles: rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes and peroxisomes 5. Describe the structure and function of the extracellular matrix 6. Describe the structure and function of the nucleus, DNA replication and the process of mitosis 7. Describe the structure and function of DNA and RNA 8. Describe how protein chains are produced under the direction of RNA via transcription and translation 5 Topic 1: Part 1 The Cell 6 CTR In Part 1, we will cover… By the end of this Topic 1, students will be able to: 1. Describe the major differences between prokaryotes and eukaryotes 2. Describe the structure and function of the plasma membrane and explain the various forms of cellular transport 3. State the components of the cytoplasm and describe the structure and function of the cytoskeleton, centrioles, cilia and flagella 4. Discuss the structures and functions of the following organelles: rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes and peroxisomes 5. Describe the structure and function of the extracellular matrix 6. Describe the structure and function of the nucleus, DNA replication and the process of mitosis 7. Describe the structure and function of DNA and RNA 8. Describe how protein chains are produced under the direction of RNA via transcription and translation 7 Prokaryotes & eukaryotes Cells are the basis of biological life – there are two distinct classes of cells, one a lot simpler in structure to the other a) PROKARYOTES without a nucleus (anuclear) and of much simpler structure generally; bacteria and archaea are prokaryotes, and were the first life forms on earth b) EUKARYOTES with a nucleus, and characterised by numerous, often complex, membrane bound inclusions; cells in all other known organisms e.g. protists, fungi, plants and animals are eukaryotes [Prokaryote – ‘before a nucleus’. Eukaryote – “possess a nucleus”] 8 While all cells have several features in common (i.e. cell membrane, require energy, store information in genes etc.) there are several major eukaryote cells. Plasma membra ne differences between prokaryote and 9 Eukaryotes vs prokaryotes ▪ There are 4 key differences between eukaryotic and prokaryotic cells: 1. Eukaryotic chromosomes are found inside a membrane bound compartment called a nucleus; prokaryotes have DNA strands in the cytoplasm 2. Eukaryotic cells are generally much larger 3. Eukaryotic cells contain extensive amounts of internal membrane, and organelles 4. Eukaryotic cells feature a diverse and dynamic cytoskeleton 10 Prokaryotes 11 How successful are the ‘primitive’ prokaryotes? ▪ Prokaryotes are microscopic, cannot be seen with the naked eye, and are studied by microbiologists ▪ However they first appeared on Earth about 3.5 billion years ago, long before eukaryotes evolved, and remain the most diverse and numerous organisms on earth, and therefore the most successful organisms, by far ▪ In fact humans have more bacterial cells living in and on them than we have cells of our own! And the numbers are in the trillions ▪ Most of these are helpful, or harmless, but some can cause disease, depending on the circumstances 12 Science Myth Busted http://www.nature.com/news/scientists-bust-myth-that-our-bodies have-more-bacteria-than-human-cells-1.19136 ▪ 1972 - Ratio of bacteria to human cells reported by Thomas Luckey as 10 bacteria: 1 human cell ▪ 2016 – Revised to 1 bacteria: 1 human cell. ▪ Total mass = 0.2 kg ▪ Sender and Fuchs (2016) Revised Estimates for the Number of Human and Bacteria Cells in the Body PLOS Biology (Aug 19) http://dx.doi.org/10.1371/journal.pbio.1 002533 13 Eukaryotes These range from single-celled organisms, e.g. algae, amoeba, fungi, yeast to multi-cellular e.g. all plants and all animals, which include sponges, insects, birds, mammals (The term ‘cell’ will refer to eukaryotic cells from this point on, and unless otherwise specified) 14 What cells are composed of? THE CELL is composed of Nucleus Cytosol Membranous organelles • Mitochondria • Endoplasmic reticulum • Golgi apparatus • Lysosomes Cytoplasm Inclusions • Lipid droplets • Glycogen granules • Ribosomes • Peroxisomes Cell membrane Protein fibers • Cytoskeleton • Centrioles • Cilia • Flagella Understand the basic structure & function of each of the listed structures 15 Extracellular fluid Eukaryotic cell structure ▪ The following structures can be identified in all eukaryotic cells*: ▪ Plasma membrane surrounding cells (some plants and fungi have a cell wall also) ▪ Cytoplasm containing: the cytoskeleton, organelles, inclusions and the cytosol. ▪ A membrane bound nucleus ▪ Intracellular material and membrane bound organelles with specific functions. With one or two exceptions; e.g. rbc lose organelles, and even their nuclei, as they mature 16 The Plasma Membrane 17 [Know all the terms listed, and be able to draw basic structure] Primary roles of the plasma membrane include: ▪ Controlling movement of water soluble substances into and out of the cell – for example sodium ions, glucose, proteins ▪ Contain receptors for signaling molecules that ‘turn on’, or ‘off’, processes in the cell ▪ Contains a ‘fuzzy coat’ of glycoproteins and glycolipids which are crucial to cell-cell attachment and cell recognition Note that there is less control over movement of fat soluble molecules because the plasma membrane is largely composed of lipids – like dissolves in like, and small fat soluble molecules usually pass unhindered 18 No biological life exists on earth in the absence of a plasma membrane ▪ Life as we know it could not have evolved without the formation of plasma membranes – the complex reactions of life must occur in a compartment that is isolated from the surrounding environment ▪ If the [concentration] of materials in a cell are ever identical to the those in the fluid that surround it, the cell is dead Use of [ ] brackets will commonly indicate concentrations 19 Structure of the plasma membrane ▪ basic structural unit of the PM is the phospholipid bilayer, a somewhat fluid and flexible structure ▪ fluidity depends on temperature, lipid composition and cholesterol content of the membrane. 20 Concept Map of Cell Membrane Components Cell Membrane consists of Cholesterol Phospholipids, Sphingolipids together form Lipid bilayer functions as together form Glycolipids Carbohydrates together form Glycoproteins whose functions include Selective barrier between cytosol and external environment Proteins Structural stability Cell recognition Immune response Ignore the term ‘sphingolipids’ for the time being, the rest of the names should be memorised. It is important to note the term ‘selective barrier’ because the main function of the PM is to be highly selective about what gets in and out of cells) 21 What makes up the cytoplasm? ▪This is the viscous region outside the nucleus containing all the organelles, cytosol, inclusions and cytoskeleton. 22 Structure of the cytoskeleton Contains filaments made of different proteins 1. microtubules (tubulin) 2. microfilaments (actin) 3. intermediate filaments (variety of proteins) Needed for 1) cell shape, 2) anchoring point 3) cell division 4) movement 23 Microtubules ▪ Microtubules are large, hollow tubes made of tubulin ▪ Microtubules provide stability and are involved in movement; they also provide a structural framework for organelles ▪ Microtubules can act as “railroad tracks” transporting vesicles and moving organelles through the cell in an energy-dependent process ▪ Animal cells have a microtubule organising centre called the centrosome. Centrosomes contain two bundles of microtubules called centrioles which direct DNA movement during cell division (see later lectures) http://www.youtube.com/watch?v=5rqbmLiSkpk 24 Microtubules & Motility Cilia and Flagella ▪ exist on cell surfaces ▪ motile elements, containing microtubules ▪ cilia beat back and forth, flagella rotate ▪ cilia can move entire cells, or move substances across cell surfaces Ciliates move by cilia ▪ flagella occur in only one cell type in the human, sperm, but are common in protists and occur in some prokaryotes 25 Actin Filaments ▪ Actin filaments are the smallest cytoskeletal elements ▪ Help determine cell shape and control of movement of structures inside cells ▪ As a result, the inside of a cell is a very dynamic place *Note: actin is also fundamental to contraction of skeletal muscles where entire groups of cells contract together 27 Actin & Motility Amoeboid movement https://youtu.be/7pR7TNzJ_pA Cytoplasmic streaming https://youtu.be/8edk6nGMwMs Memorising the details not required 28 Intermediate Filaments ▪ Intermediate filaments are defined by size rather than composition. Many types of intermediate filaments exist, each consisting of a different protein https://youtu.be/ll5MSxxHSCQ ▪ Intermediate filaments provide structural support for the cell. They are not involved in movement ▪ Intermediate filaments form a flexible skeleton that helps shape the cell surface and hold the nucleus in place Notes added for completion, but the role of these filaments will not be stressed or discussed much subsequently. 29 Compartmentalization within cells ▪ As with the requirement for plasma membranes to separate the intra-cellular compartment from the external environment, so it is within the cell itself ▪ Complex and diverse function can only exist within cells when specialized compartments isolate specialized material from the rest of the cell discrete membrane bound compartments are termed organelles; the nucleus is also membrane bound 30 Compartmentalization ▪ The compartmentalization of eukaryotic cells allows packaging of materials within the cell… ▪ Thereby permitting metabolic reactions to occur independently of the rest of the cell cytoplasm and cell machinery ▪ Allowing specific products to be exported from cells ▪ Allowing isolation of products that are dangerous to the cell itself, such as digestive enzymes 31 The Nucleus 32 Structure & function of the nucleus STRUCTURE ▪ double membrane nuclear envelope ▪ pores allow movement of RNA and proteins FUNCTIONS ▪ houses genetic material as chromosomes ▪ nucleolus: rRNA synthesised and ribosome subunits assembled here 33 Nucleus – electron microscopy For illustration only, details of micrographs not required 34 Endoplasmic reticulum 35 1. Rough Endoplasmic Reticulum ▪ STRUCTURE: ▪ The rough endoplasmic reticulum (rough ER) is a network of membrane bound tubes and sacs studded with ribosomes ▪ Rough ER is continuous with the nuclear envelope ▪ FUNCTION: ▪ Ribosomes associated with the rough ER synthesize proteins ▪ New proteins are folded and processed in the rough ER lumen. 36 37 Ribosomes: protein factories ▪ STRUCTURE: ▪ Ribosomes are non membranous (so not truly organelles, but are associated with them) ▪ Have large and small subunits, both containing RNA molecules and protein ▪ Ribosomes can be attached to the rough ER or exist free in the cytosol, the fluid part of the cytoplasm ▪ FUNCTION: ▪ Protein synthesis 38 2. Smooth Endoplasmic Reticulum ▪ STRUCTURE: ▪ The smooth endoplasmic reticulum (smooth ER) lacks the ribosomes associated with the rough ER, and so lack the ‘rough’ bumps of ribosomal ER; it is continuous with the rough ER but plays no part in protein synthesis ▪ FUNCTIONS include: ▪ Metabolise and synthesis of lipids, cholesterol ▪ Absorb and transport lipids obtained from GI tract ▪ Posses enzymes which can detoxify substances such as pesticides ▪ Reservoir for Ca2+ ions 39 40 Golgi Apparatus 41 Golgi Apparatus STRUCTURE: ▪ The Golgi apparatus is formed by a series of stacked flat membranous sacs, that pinch off to form enclosed spherical containers, termed vesicles FUNCTIONS: ▪ Major role is to modify, concentrate and package proteins and lipids made in the ER ▪ These products when packaged in vesicles are sent to the plasma membrane for export, or to other organelles within the cell 42 Mitochondria It is now widely accepted that mitochondria have prokaryote origins, were subsumed in a cell line billions of years ago and once this happened allowed for the burning of oxygen in what is now known as aerobic respiration – what advantage did this confer? 43 Mitochondria STRUCTURE: ▪ Mitochondria have two membranes; the inner one is folded into a series of sac-like cristae. Between the cristae is the mitochondrial matrix ▪ Mitochondria have their own DNA and manufacture their own ribosomes FUNCTION: ▪ ATP production is the mitochondrion’s core function 44 The origins of mitochondria ▪ To reiterate, there is little doubt that mitochondria derive from a prokaryote cell that learnt to burn oxygen and was then ‘ingested’ by another, and set to work ▪ Mitochondria allow for the ‘burning’ of a glucose to produce about 38 molecules of ATP (or energy, essentially) per molecule of glucose, compared to 2 when oxygen cannot be so utilized ▪ This astonishing increase in efficiency allowed the larger and more complex eukaryotes to evolve – all complex multicellular organisms, like us, owe their success to a captured prokaryote! 45 Lysosomes The demolition crew of the cell, disposing of the unwanted from ▪ within the cell, e.g. old organelles ▪ outside the cell, e.g. bacteria, and ▪ entire unwanted cells of the body – cell suicide 46 Lysosomes STRUCTURE: ▪ Lysosomes are single-membrane bound structures containing approximately 40 different digestive enzymes FUNCTIONS: ▪ Lysosomes can digest/ denature almost all biological macromolecules, they help dispose of bacteria, defective/old cells and organelles etc, and allow calcium to be released (digested) from bone, for example Note : if lysosomes rupture following cell damage, cells digest themselves – this is 47 termed autolysis Lysosomes 48 Peroxisomes 49 Peroxisomes STRUCTURE: ▪ Peroxisomes are spherical organelles bound by a single membrane. FUNCTION: ▪ contain enzymes to degrade harmful substances, most importantly to neutralise oxygen free radicles, converting these to hydrogen peroxide ▪ H2O2 is then degraded to water and O2 50 Cytoplasm – inclusions ▪ Inclusions: substances not membrane bound i.e. glycogen granules, lipid droplets in adipose tissue cells ▪ many enzymes, large proportion of total cell protein, ions, metabolic intermediates 51 http://missinglink.ucsf.edu/lm/IDS_101_histo_resource/epithelia_connective.htm Cellular links to the extracellular matrix- ECM Ground substance is unstructured material that exists between cells. It contains: ▪ cell-adhesion molecules, such as lamelin, that hold/adhere cells to connective tissue fibres ▪ proteoglycans (sugar units linked to a protein core, split term to understand it); these help form the ‘fuzzy’ cell coat 52 Cellular Transport The endomembrane system is comprised of organelles involved in production, storage and export of molecules, and degradation of potentially harmful substance ▪ i.e. it regulates protein traffic and breakdown of molecules in the cell Components of the endomembrane system include: ▪ ER, Golgi apparatus, the nuclear envelope, lysosomes, vacuoles vesicles; the plasma membrane also plays a role ▪ Proteins move from RER to Golgi apparatus; proteins with distinctive molecular labels are then shipped to where-ever in the cell they are required ▪ Proteins for export are enclosed in vesicles and sent to the plasma membrane; when vesicular membrane fuses with the plasma membrane, the proteins are released into the extra cellular compartment 53 Cellular Transport Nucleus: ▪ Messenger RNAs and ribosomal subunits are synthesised in the nucleus and exported to the cytoplasm via nuclear pores in the nuclear envelope ▪ Materials such as proteins needed in the nucleus are imported into the nucleus 54 Cellular Transport 55 Cellular Transport ▪ The cytoskeleton is a complex network of fibres that helps maintain cell shape by providing structural support ▪ The cytoskeleton is dynamic; it changes to alter the cell’s shape, to transport materials in the cell, or to move the cell itself. https://youtu.be/297HcgDxb7k 56 Topic 1 Readings ▪ Part 1: Read Sections 3.1- 3.2 and 3.7- 3.8 of Chapter 3 Cells: The Living Units (10th/11th Ed) ▪ Part 2: Read Sections 3.9- 3.10 of Chapter 3 Cells: The Living Units (10th/11th Ed) For revision on DNA and RNA structure, see Section 2.11 in Chapter 2 Chemistry Comes Alive (10th/11th Ed) ▪ Part 3: Read Sections 3.11 of Chapter 3 Cells: The Living Units (10th/11th Ed) 57 In Part 2, we will cover… By the end of this Topic 1, students will be able to: 1. Describe the major differences between prokaryotes and eukaryotes 2. Describe the structure and function of the plasma membrane and explain the various forms of cellular transport 3. State the components of the cytoplasm and describe the structure and function of the cytoskeleton, centrioles, cilia and flagella 4. Discuss the structures and functions of the following organelles: rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes and peroxisomes 5. Describe the structure and function of the extracellular matrix 6. Describe the structure and function of the nucleus, DNA replication and the process of mitosis 7. Describe the structure and function of DNA and RNA 8. Describe how protein chains are produced under the direction of RNA via transcription and translation 58 Topic 1: Part 2 Cell & DNA replication 59 CTR Nucleic Acids ▪ Make up DNA and RNA ▪ Largest molecules in the body ▪ Contain carbon (C), oxygen (O), hydrogen (H), nitrogen (N), and phosphate (P) ▪ Basic building blocks are nucleotides, composed of: N-containing base, a pentose sugar, and a phosphate group 60 Deoxyribonucleic Acid (DNA) ▪ Four bases adenine (A), guanine (G), cytosine (C), & thymine (T) ▪ Double-stranded helical molecule in the cell nucleus ▪ Provides instructions for protein synthesis ▪ Replicates before cell division, ensuring genetic continuity Note: while there are only 4 bases, this short ‘alphabet’ allows for an almost infinite number of different proteins to be formed; this will be expanded on subsequently 61 Phosphate Base: Adenine (A) Sugar: Deoxyribose Phosphate Thymine (T) Sugar Adenine nucleotide (a) Sugar-phosphate backbone Deoxyribose sugar Phosphate Adenine (A) Thymine (T) Cytosine (C) Guanine (G) (b) Thymine nucleotide Hydrogen bond (c) Computer-generated image of a DNA molecule 62 Ribonucleic Acid (RNA) ▪ Four bases, with one change, uracil for thymine: ▪ adenine (A), guanine (G), cytosine (C), uracil (U) ▪ Single-stranded molecule mostly active outside the nucleus ▪ Three varieties of RNA carry out the DNA orders for protein synthesis ▪ messenger RNA, transfer RNA, and ribosomal RNA 63 Comparison of DNA and RNA 64 65 Organization of the DNA strands in the nucleus Be able to recall • The helical nature of DNA • What chromatin is • Role of histone proteins • What a chromatid is Chromatin ▪ Visible with light microscopy ▪ 30% DNA ▪ 60% histone proteins which package & regulate DNA ▪ 10% RNA chains (newly formed or forming) Role of Histones ▪ Allow packaging of extremely long DNA strands (2m per cell!) ▪ Shut down DNA when needed ▪ Activate DNA segments to allow protein production 66 Chromatin to chromosomes ▪ Prior to cell division, chromatin threads condense to form very compact, short ‘bar’ structures ▪ These are called chromosomes ▪ The DNA strands in this form are stable and not easily disrupted as they move during cell division Source: https://www.yourgenome.org/facts/what-is-a chromosome 67 Cell division ▪ Mitosis is duplication of chromosomes, with one set being given to 2 new daughter cells – this will discussed further ▪ Meiosis is a process by which the chromosome number is halved in daughter cells; these become the gametes which when combined in sexual reproduction give the fertilized egg a full chromosome complement again – meiosis will not be discussed further in this course 68 Cell Cycle 69 During the S-interphase, cells grow and DNA is synthesised and replicated – for cells that will not divide (and there are some), their entire life is spent in the early G1-interphase stage 70 DNA Replication ▪ DNA helices begin unwinding ▪ Helicase (an enzyme) untwists the double helix and exposes complementary chains ▪ The Y-shaped site of replication is the replication fork ▪ Each nucleotide strand serves as a template for building a new, complementary strand 71 DNA Replication ▪ DNA polymerases only work in one direction to position complementary free nucleotides next to those in the DNA strand, and then acts to covalently bond these, forming new double helixes ▪ The leading strand is synthesized continuously ▪ The lagging strand (moving in the opposite direction) is constructed in segments; ligase enzymes splice the short DNA segments together 72 73 DNA Replication ▪ End result: two DNA molecules formed from the original https://youtu.be/OjPcT1uUZiE?t=1m43s 74 Chromatin condenses to form chromosomes, which are visible with light microscopy. The centromeres hold the paired chromosomes together. The centrosomes are involved in formation of microtubules which form the mitotic spindle. 75 Now the nuclear membrane breaks down, and now the spindle can interact with the chromosomes. The duplicated chromosomes consist of two identical threads termed sister chromatids 76 The centrosomes are now at opposite poles of the cell, and chromosomes are aligned at the equator 77 The 3rd and shortest phase: the chromosomes spit, and a complete set of chromosomes are pulled apart to move toward the poles 78 This is essentially prophase in reverse; a nuclear envelop forms around a complete set of chromosomes which then unwind and form ‘functional’ threads of chromatin; the spindle breaks down, and until the cell contains 2 nuclei for the short period before the cell membranes spans the cleavage furrow and 2 complete daughter cells come into existence again 79 Topic 1 Readings ▪ Part 1: Read Sections 3.1- 3.2 and 3.7- 3.8 of Chapter 3 Cells: The Living Units (10th/11th Ed) ▪ Part 2: Read Sections 3.9- 3.10 of Chapter 3 Cells: The Living Units (10th/11th Ed) For revision on DNA and RNA structure, see Section 2.11 in Chapter 2 Chemistry Comes Alive (10th/11th Ed) ▪ Part 3: Read Sections 3.11 of Chapter 3 Cells: The Living Units (10th/11th Ed) 80 Topic 1: Part 3 Protein Production 81 CTR In Part 3, we will cover …. By the end of this Topic 1, students will be able to: 1. Describe the major differences between prokaryotes and eukaryotes 2. Describe the structure and function of the plasma membrane and explain the various forms of cellular transport 3. State the components of the cytoplasm and describe the structure and function of the cytoskeleton, centrioles, cilia and flagella 4. Discuss the structures and functions of the following organelles: rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes and peroxisomes 5. Describe the structure and function of the extracellular matrix 6. Describe the structure and function of the nucleus, DNA replication and the process of mitosis 7. Describe the structure and function of DNA and RNA 8. Describe how protein chains are produced under the direction of RNA via transcription and translation 82 Life is all about proteins ▪ Proteins run and control life processes, involved in most everything imaginable and crucial to life – for example, enzymes, receptors, contractile muscles fibres, immuno globulins are proteins….. ▪ The DNA code determines protein production; DNA is the master blueprint for protein synthesis ▪ Genes code for proteins; a genetic mutation means a change in an amino acid in a protein chain ▪ Triplets of nucleotide bases form the genetic library; these triplets code for amino acids ▪ How DNA directs the production of proteins is now to be discussed 83 Proteins and amino acids remember that…. ▪ each protein is composed of long, folded chains of numerous, often hundreds of, linked amino acids ▪ the nature and position of each amino acid in that chain is determined by genes ▪ a genetic mutation causes a change in a single aa in that chain; this may have no effect on function of the protein, or it may in fact ‘ruin’ it entirely 84 DNA to protein production Always remember : ▪ DNA holds the code, but this needs to be translated to effect protein production outside the nucleus ▪ DNA nucleotides determine RNA sequences ▪ RNA takes the code to the cytoplasm 85 Transcription and translation – overview Transcription RNA Processing mRNA Translation DNA Nuclear envelope Pre-mRNA Nuclear pores Ribosome Polypeptide 86 3 Main Types of RNA 1. Messenger RNA (mRNA) ▪ carries instructions for building a polypeptide; the instructions are determined by a gene in the DNA and taken to ribosomes in cytoplasm 2. Transfer RNA (tRNA) ▪ Each tRNA has an aa-binding sited, and a codon binding site, allowing binding to complementary codons in mRNA ▪ Sequential tRNA codon binding to mRNA allows aa’s to be added to an aa-chain, to begin process of protein synthesis ▪ The linking of codons at one end, and additoin of aa’s at the other, is directed by ribosomes 3. Ribosomal RNA (rRNA) is linked to the ribosomal proteins, and assists in tRNA linking of amino acids not discussed further at this stage 87 Nucleoli ▪ The ‘little’ nuclei, usually 1 or 2 only per cell ▪ These organize the transfer of DNA instructions to the ribosomal RNA ▪ Ribosomal proteins move into the nucleus from the cytoplasm, RNA is attached to them and the complexes are then move into the cytoplasm, via nuclear pores 88 DNA to RNA to proteins overview ▪ DNA provides the basic code for protein production, but DNA never leaves the nucleus ▪ However, protein production occurs outside the nucleus ▪ In essence, the DNA code must be decoded and sent out in a form that can direct protein production ▪ The decoded DNA messages are carried by RNA, and together with ribosomes, protein production occurs 89 Transcription ▪ Transfer of DNA gene base sequence to a complementary base sequence of an mRNA ▪ Transcription factor ▪ Loosens histones from DNA in area to be transcribed ▪ Binds to promoter, a DNA sequence specifying start site of gene to be transcribed ▪ Mediates the binding of RNA polymerase to promoter 90 Transcription ▪ RNA polymerase ▪ Enzyme that oversees synthesis of mRNA ▪ Unwinds DNA template ▪ Adds complementary RNA nucleotides on DNA template and joins them together ▪ Stops when it reaches termination signal ▪ mRNA pulls off the DNA template, is further processed by enzymes, and enters cytoplasm 91 RNA polymerase DNA 1 Promoter region Coding strand Template strand Termination signal Initiation: With the help of transcription factors, RNA polymerase binds to the promoter, pries apart the two DNA strands, and initiates mRNA synthesis at the start point on the template strand. 92 2 mRNA Template strand Elongation: As the RNA polymerase moves along the template strand, elongating the mRNA transcript one base at a time, it unwinds the DNA double helix before it and rewinds the double helix behind it. mRNA transcript 93 Coding strand of DNA Rewinding of DNA mRNA RNA nucleotides Direction of transcription DNA-RNA hybrid region Unwinding of DNA Template strand RNA polymerase The DNA-RNA hybrid: At any given moment, 16–18 base pairs of DNA are unwound and the most recently made RNA is still bound to DNA. This small region is called the DNA-RNA hybrid. 94 3 Termination: mRNA synthesis ends when the termination signal is reached. RNA polymerase and the completed mRNA transcript are released. Completed mRNA transcript RNA polymerase 95 Translation – whereby mRNA and tRNAdetermine protein production ▪ mRNA attaches to a small ribosomal subunit that moves along the mRNA to the start codon ▪ The large ribosomal unit attaches, forming a functional ribosome ▪ Anticodon of a tRNA binds to its complementary codon and adds it’s amino acid to the forming protein chain ▪ New amino acids are added by other tRNAs as the ribosome moves along rRNA, until the stop codon is reached https://youtu.be/5bLEDd-PSTQ 97 Nucleus mRNA 1 After mRNA synthesis in the nucleus, mRNA leaves the nucleus and attaches to a ribosome. 2 Translation begins as incoming aminoacyl-tRNA recognizes the complementary codon calling for it at the A site on the ribosome. It hydrogen-bonds to the codon via its anticodon. 3 As the ribosome moves along the mRNA, and each codon is read in sequence, a new amino acid is added to the growing protein chain and the tRNA in the A site is translocated to the P site. RNA polymerase Template strand of DNA Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Leu Amino acid Nuclear pore tRNA Nuclear membrane Released mRNA Ile Pro Leu G A A tRNA “head” bearing anticodon 4 Once its amino acid is released from the P site, tRNA is ratcheted to the E site and then released to reenter the cytoplasmic pool, ready to be recharged with a new amino acid. The polypeptide is released when the stop codon is read. Figure 3.37 Portion of mRNA already translated E site P site G G A U A Codon 15 C C C Codon 16 Direction of ribosome advance A site C G U U Codon 17 Large ribosomal subunit Small ribosomal subunit Aminoacyl-tRNA synthetase 98 Translation – first steps Nucleus mRNA 1 After mRNA synthesis in the nucleus, mRNA leaves the nucleus and attaches to a ribosome. RNA polymerase Template strand of DNA Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl tRNA synthetase enzyme. Leu Amino acid Nuclear pore tRNA Nuclear membrane Released mRNA G AA Aminoacyl-tRNA synthetase Crucial point: amino acid Leucine (Leu) is about to be attached to a tRNA segment 99 Leu Ile Pro P site E site G G tRNA “head” bearing anticodon C A site Large ribosomal subunit A U A Codon 15 Portion of mRNA already translated C C C G U U Codon 16 Codon 17 Direction of ribosome advance Small ribosomal subunit 2 Translation begins as incoming aminoacyl-tRNA recognizes the complementary codon calling for it at the A site on the ribosome. It hydrogen-bonds to the codon via its anticodon. mRNA and tRNAnucleotides pair up, and the latest aa (Pro) linked to its specific tRNA, has now been attached to the ‘front’ of the developing amino acid chain; Leu will be next (see C U U codon in A site – this codon moves to the P site as the ribosome advances to the right) 100 Leu 3 As the ribosome moves along the mRNA, and each codon is read in sequence, a new amino acid is added to the growing protein chain and the tRNA in the A site is translocated to the P site. 4 Once its amino acid is released from the P site, tRNA is ratcheted to the E site and then released to reenter the cytoplasmic pool, ready to be recharged with a new amino acid. The polypeptide is released when the stop codon is read. Ile Pro tRNA “head” bearing anticodon 2 E site P site G G C A site Large ribosomal subunit A U A Codon 15 Portion of mRNA already translated C C C G U U Codon 16 Codon 17 Direction of ribosome advance Small ribosomal subunit Translation begins as incoming aminoacyl-tRNA recognizes the complementary codon calling for it at the A site on the ribosome. It hydrogen-bonds to the codon via its anticodon. 101 Polyribosome arrays Growing polypeptides Incoming ribosomal subunits Start of mRNA Polyribosome Completed polypeptide (a) Each polyribosome consists of one strand of mRNA being read by several ribosomes simultaneously. End of mRNA In this diagram, the mRNA is moving to the left and the “oldest” functional ribosome is farthest to the right. Ribosomes mRNA (b) This transmission electron micrograph shows a large polyribosome (400,000x). 102 Protein processing by rough ER 1 2 The mRNA-ribosome complex is directed to the rough ER by the SRP. There the SRP binds to a receptor site. Once attached to the ER, the SRP is released and the growing polypeptide snakes through the ER membrane pore into the cisterna. ER signal sequence Signal recognition particle (SRP) Receptor site Growing polypeptide Rough ER cisterna 3 Ribosome mRNA The signal sequence is clipped off by an enzyme. As protein synthesis continues, sugar groups may be added to the protein. Signal sequence removed Sugar group 4 In this example, the completed protein is released from the ribosome and folds into its 3-D conformation, a process aided by molecular chaperones. Released protein Cytoplasm 5 The protein is enclosed within a protein (coatomer)-coated transport vesicle. The transport vesicles make their way to the Golgi apparatus, where further processing of the proteins occurs (see Figure 3.19). Transport vesicle pinching off Coatomer-coated transport vesicle 103 105 DNA to proteins - review ▪ DNA is the master blueprint for protein synthesis and a gene is segment of DNA with a blueprint for one particular polypeptide ▪ Each three-base sequence on DNA is represented by a matching/complementary three-base sequence on mRNA, termed a codon ▪ Transcription describes this transfer of information to mRNA ▪ Once in the cytoplasm the mRNA message is translated to produce aa chains via interactions of mRNA, tRNA and ribosomes (themselves proteins); each mRNA codon has a matching tRNA anticodon ▪ Remember: translation converts base sequence of nucleic acids into the amino acid sequence of proteins 106 Topic 1 Readings ▪ Part 1: Read Sections 3.1- 3.2 and 3.7- 3.8 of Chapter 3 Cells: The Living Units (10th/11th Ed) ▪ Part 2: Read Sections 3.9- 3.10 of Chapter 3 Cells: The Living Units (10th/11th Ed) For revision on DNA and RNA structure, see Section 2.11 in Chapter 2 Chemistry Comes Alive (10th/11th Ed) ▪ Part 3: Read Sections 3.11 of Chapter 3 Cells: The Living Units (10th/11th Ed) 107