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PDFs of all lectures available from my teaching webpage Lecture 1: What is inside the cell? 1. The flow of genetic information: “The Central Dogma of Molecular Biology” 2. What is inside the cell? - From g genes to proteins: p - Nucleus - Endoplasmatic reticulum - Ribosomes - Golgi complex - Mitochondria - Nucleolus Sergey Kasparov Room E9 [email protected] THE CENTRAL DOGMA Genes (parts of DNA) Intermediates (messenger RNA) Proteins Prokaryotic versus Eukaryotic cells Prokaryotic Eukaryotic Cells lacking a membranebound nucleus are called prokaryotes. From the Greek meaning “before nuclei” A eukaryotic cell has the genetic material organized into a membrane bound nucleus Nucleus contains the genetic code. Cell = nucleus + cytoplasm. Cytoplasm = cytosol + organelles. 1 e.m. 1. 2. 3. 4. Nuclear envelope (2 layers of membrane) Nuclear pores (tightly controlled gates!) Chromatin (loose strings of DNA, the genetic material) Nucleolus Nuclear membrane is a continuation of the “endomembrane”. Endoplasmatic reticulum What is chromatin? e.m. Laemmli DNA – the molecule which contains the genetic code (which is a collection of recipes for proteins!). Between the divisions it needs to be loosely spread to allow access to its various parts. 2 Nuclear membrane has pores: Pores are not just “holes”! Nuclear membrane These are specialised micro-channels which are highly selective and allow traffic of specific molecules from the nucleus and into the nucleus. Nuclear pores visualised by the freeze-fracture technique Outer membrane The genetic instructions (recipes) stay inside the nucleus 3 Production of mRNAs is a tightly controlled process Signalling molecules 1. There is a continuous exchange of information between the nucleus and cytoplasm. 2. Specialised proteins come into nucleus, nucleus they bind to DNA to regulate production of specific messenger RNAs (mRNAs). DNA Messenger 3. Messenger RNAs pass molecules from nucleus into the cytoplasm. They encode proteins which determine i) cell structure ii) cell function. Outside of the nucleus: the endoplasmatic reticulum Endoplasmatic reticulum Rough ER (granular) Smooth ER (agranular) 4 Two types of Endoplasmatic reticulum Rough (or granular) endoplasmatic reticulum Organelle which makes the ER “rough”: The ribosome Small subunit 40S Large subunit 60S 1. Ribosomes consist of 2 subunits (parts) of different sizes. 2. Chemically ribosomes are comprised of a special type of RNA (rRNA) and a number of small proteins. 3. They synthesise proteins according to the sequence of the messenger molecules (mRNA) which arrive from nucleus. 5 Point of Interest: Ribosomes of bacteria and mammalian cells are different in their composition. Because of that antibacterial drugs (antibiotics) may selectively block protein synthesis p y in bacteria and kill them. Summary so far The key points: 1. The messenger (mRNA) molecule arrives from the nucleus and acts as a template. 2. Subunits of the ribosome “embrace” this template and act as a docking station for the arriving building protein (aminoacids) ( ) blocks of the p 3. The sequence of these blocks is encoded by the messenger (mRNA) 4. Ribosomes of bacteria and mammalian cells are different in their composition. Components of ribosomes are produced in the nucleus by the structure known as the nucleolus 6 Proteins which need to be secreted out of the cell are directed from R-ER into the Golgi apparatus (Golgi complex) Rough ER (granular) Smooth ER (agranular) Smooth (or agranular) endoplasmatic reticulum 7 Smooth (agranular) ER: 1. Tubular network that does not have ribosomes attached to it. 2. Functions: • A. Contains machinery y for production p of certain molecules (i.e. lipids) • B. Stores and releases calcium ions, which control various cell activities, for example contraction of (cardiac) muscle cells Mitochondria (singular: mitochondrion) The key points: 1. Mitochondria are numerous small organelles < 1μM in size. They are the major site of cell energy production from ingested nutrients. 2. This process involves oxygen consumption and CO2 formation. formation It leads to formation of ATP (adenosine triphosphate) – the fuel of the cell. 3. Cells which utilize large amounts of energy contain as many as 1000 of them. 4. Have two layers of membrane. The inner layer forms “cristae” –membrane folds to increase the inner surface area. 5. May replicate independently of the host cell. 8 The origin of mitochondria: It is thought that mitochondria have originated from bacteria which have “learned” to live inside of their host cells permanently and be useful, rather than harmful. Salmonella bacterium Evolution of cells Additional interesting facts: 1. Mitochondria have their own DNA, which replicates independent of the nuclear DNA 2. Genetic code of the mitochondria is different from the main code of the cell 3. Mitochondria have their own ribosomes on which some of the mitochondrial proteins are produced. Others are imported from the outside 4. There are genetic disorders which are due to mutations in mitochondrial genes 5. We inherit our mitochondria from mothers because sperms only release their DNA during fertilisation 9 Cytoskeleton component Diameter Building blocks (protein monomers) Examples of function Microfilament 7 nm G-Actin Ubiquitous component of cytoskeleton, Contractile protein of skeletal muscles. m . Microtubule 25 Tubulin Support membrane protrusions. Act as intracellular “railways”. Separate chromosomes during cell division. DNA - RNA - Protein Nucleus: contains DNA, the source of the genetic code. It is responsible for storage of the code and its retrieval (e.g. production of mRNAs for the proteins required by the cell) Ribosomes: assemble proteins according to the instructions relayed by the messenger RNA Nucleolus – a dense spot p in the nucleus where the rRNA required for production of ribosomes is produced Rough Endoplasmatic Reticulum: provides the scaffolding for the ribosomes and is involved in protein maturation Golgi complex: a system of cisterns involved in protein sorting and traffic Mitochondria: supply fuel (ATP) for all cellular activities, including genome function 10 PDFs of all lectures available from my teaching webpage Lecture 2 THE CENTRAL DOGMA OF MOLECULAR BIOLOGY: The Universal Genetic Code Information Transfer From DNA-to- mRNA – to - Protein 11 States of DNA Chromosomes – supercoiled DNA just before cell division DNA – the molecule which contains the genetic code. Between the divisions it is loosely spread in the nucleus which is essential to allow access to its various parts. Nucleotides: the letters of the genetic code Components of a nucleotide: a) A sugar Found in RNA Found in DNA Nucleotides: the letters of the genetic code Components of a nucleotide: a) A sugar b) A residue of phosphoric acid c) One of the bases O -O-P-O 12 H DNA is built of numerous nucleotides connected via phosphoric acid residues DNA is a linear molecule and it has a “beginning” (5’ end with phosphoric acid residue attached) and “end” (3’ end with free sugar OH group). H backbone ? TOO FAR ? E. Chargaff’ law: the amount of adenine is always equal to that of thymine, and the amount of guanine to that of cytosine. A & T and C & G form complimentary pairs. Thus, for each sequence of nucleotides there is a complimentary sequence. 13 DNA: the double helix MOVIE Why go double stranded –1 ? Double-stranded conformation has very low potential energy (e.g. is the most thermodynamically favourable state). Why go double stranded –2 ? Always keep a back-up copy: in case one of the strings gets damaged damaged, the other can be used as a template to restore the information: 14 It is possible to break the two strings apart by heating the DNA. This process is often called “melting”. However when DNA cools down the two strings will re-anneal! Heat Cool Points to NOTE: 1. Sequences with high CG (3 bonds) content are much more difficult to break apart than those with high AT content (2 bonds). How can we guess the GC content of a DNA sample from its melting temperature? 2. RNA and DNA may anneal to each other. This is called hybridisation and is used in some experimental techniques. 3. DNA contains thymine and RNA uracil, but their role are essentially ss ti ll id identical ti l 4. It may happen that only some parts of the molecules (RNA & DNA) are complementary to each other. They will anneal to each other but non-complimentary parts of the molecules will stay separate. complimentary non-complimentary How can this system be used to encode proteins??? 15 1. Hypothesis of DNA-protein co-linearity: DNA sequence in some way describes the sequence of aminoacids DNA (a sequence of bases) PROTEIN (a sequence of aminoacids) 2. It is known that there are ~20 aminoacids How many nucleotides need to combine to encode 20 aminoacids??? There are 4 nucleotides available 41 = 4 amino acids 42 = 16 amino acids 43 = 64 amino acids 3-nucleotides are needed to make one “codon” DNA PROTEIN Mutation –1 : ONE nucleotide added PROTEIN Mutation –2 : TWO nucleotides added PROTEIN Mutation –3 : THREE nucleotides added PROTEIN 16 Triplets of nucleotides in DNA encode individual aminoacids T in DNA T in DNA T in DNA T in DNA T in DNA T in DNA Essential features of the code: 1. Linearity 2. Redundancy 3. “Wobble”: the 3rd base is the least important 3 S 3. Specialised i li d codons: d - Initiation codon (ATG – methionine) - Stop codons which do not encode any amino acid -!!!!!!!!! 4. Genetic code is UNIVERSAL (e.g. nearly the same in plants, bacteria, humans, insects etc) 17 Genetic code may be used for prediction of possible proteins based on DNA sequence analysis etc … approx. 5000 bp ATG NOTE: Only one DNA strand contains the information about the sequence of the protein. This is called the “sense” strand. The other strand (“antisense”) is just its mirror image. In order to reveal protein sequence one needs to know the sequence of the “sense” strand). TA Computers automatically detect all possible “open reading frames”. Usually it is possible to guess which one is the right one based on its length. Example: protein which might be encoded by this RNA has a molecular mass which corresponds to ~ 1700 amino acids. The gene has to have an ORF of ~5100 base pairs. 18 Mutations: accidental alterations of the code No shift in the reading frame: only a single codon altered Possible results of a “substitution” mutation: Original: AGG TAG CGA CTT Arg Trp Arg Leu AGG TAT CGA CTT Arg Cys Arg Leu AGG TAA CGA CTT Arg Consequences of missense mutations at the level of protein will depend on the nature of substitution (i.e. which amino acid replaces which). Substitutions may be “silent” (especially if it is the 3rd base in a codon). Mutations: accidental alterations of the code Shift in the reading frame: Everything past this point will be different! 19 Gene expression DNA – mRNA - PROTEIN Each cell is producing its individual set of proteins. Signalling molecules Specialised proteins (transcription bind to DNA to regulate production d i of f specific ifi messenger RNA (mRNA). factors) Genes which actively generate mRNA are “EXPRESSED”. Messenger molecules DNA Summary 9 The information in DNA is stored in codons (triplets of nucleotides) and is read linearly. A shift in reading frame will completely change the whole message. 9 From the sequence one may make guesses about proteins which it might encode 9 Mutations are “unauthorised” unauthorised alterations of the code. They do not always have visible consequences and may be beneficial or lethal. 9 Knock-out animals bare artificially introduced inactivating mutations in genes which scientists are interested in. 9 Genes may be dormant or “expressed” – this is when they start producing numerous molecules of mRNA and protein. 20 PDFs of all lectures available from my teaching webpage Lecture 3 The road from information to function: 1 Transcription Tr nscripti n 2 RNA processing 3 Translation 4 Posttranslational processing of proteins Mendel’s inheritable factors – genes. Definition of a gene? -a unit of heredity, it controls a single inheritable characteristic characteristic. - a DNA sequence which encodes for a single polypeptide chain or a single mRNA. Essential features of the code: Make sure you have this information! 1. Each “word” consists of 3 “letters” and is called a “codon” 2. There are no gaps between the “words”. Therefore if the “frame” of reading is shifted the whole sense changes 3. The code is linear: the sequence of nucleotides in the DNA is read in one direction from the beginning (5’ end) to the end (3’ end) 4. The code is redundant (e.g several words mean the same thi ) thing) 5. It “wobbles”: the 3rd base (3rd letter in each word) is the least important 6. It has specialised codons : - The initiation codon (ATG – methionine) - Stop codons which do not encode any amino acid 7. Genetic code is UNIVERSAL (e.g. nearly the same in plants, bacteria, humans, insects etc) 8. In DNA the letters of the code are: ACTG. In RNA the letters of the code are ACUG 21 ATG TA BASIC FLOW OF INFORMATION IN A CELL: DNA – RNA – PROTEIN Structure of a gene PROTEIN 1. Exons 2. Introns Only a few percent of human genome are currently thought to be exons! The genome contains cryptic messages! ELE XGTIEN PHA EMRTI NT ELE XGTIEN PHA EMRTI NT ELEPHANT PROTEIN 22 Primary RNA transcript 1 RNA SPLICING 2 3 Mature RNA Contains sequences from the exons ONLY! How does the transcription machinery work? 5’ end of the “sense” strand RNA polymerase gene In order for transcription to begin DNA must unwind and form a “fork” so that RNA polymerase can get attached to it. Promoter sequences 5’ Transcription factors RNA polymeraseII Coding part of the gene 1. Promoter sequences are located upstream of the gene 2. Specific proteins known as transcription factors can bind to these sequences and facilitate transcription 23 regulatory gene(s) RNA polymerase PROMOTER regulatory gene(s) the gene Would you like to make an entry into your handouts? RNA polymerase PROMOTER structural gene Mature mRNA 1 5’ end 2 Information Information 3 Poly-A tail 3’ end Think: What would happen if this mRNA was hybridised with its parent genomic DNA? 24 Important: The code in the mature mRNA is identical to that in the exons of the sense (coding) strand of DNA. Thus, mRNA can deliver correct information from the gene to the ribosome where protein synthesis will occur. Translation: The process of synthesis of proteins according to the information delivered by mRNA Amino acids may have very different properties: 1. 2. 3. 4. Size Charge Solubility in water and lipids Ability to form bonds with other amino acids and various molecules 25 Key points: mRNA arrives with a “cap” and a poly A tail The ribosome scans the mRNA from 5’ end to find the first methionine codon Translation is then initiated and more and more amino acids arrive attached to the tRNAs When the ribosome reaches a stop codon, translation is terminated and the new peptide chain dissociates from the ribosome The process will repeat until mRNA looses its poly-A tail and gets degraded Posttranslational Protein Modifications (Maturation) 1. Removal of certain parts of the protein molecule 2. Addition of some residues (sugars & lipids) to specific amino acids in the new protein 3. Formation of intra-protein bonds Traffic of new proteins from the ER to the Golgi complex for further processing and (in some cases) secretion from the cell What have we done… 1. Revised the structure of the cell and its basic machinery for storage of genetic information and protein production 2. Got familiar with the “Central Dogma” of molecular biology 3. Looked at the Universal Genetic Code and its li ti tto th the genomic i d data, t such h as DNA and d application RNA sequences 4. Discovered how the information stored in DNA is transferred to RNA and then used to determine the structure and functional properties of proteins 5. … and how proteins “mature”… 26 PDFs of all lectures available from my teaching webpage Lecture 4 Wonders of Molecular Biology Nuts and bolts of DNA manipulation p Transferring genes from jellyfish and coral into brain nerve cells “Wonders of Molecular Biology” 27 Why manipulate the DNA? Because we want to understand how the system works in health and disease - Find an abnormality responsible for a disease - Generate an animal model to study a disease - Search for the drug target(s) or screen the chemical molecules for their ability to bind to this target - Establish new research methods with better resolution and relevance to human diseases Tweak it Get it Put it back in Rat brain neurones made fluorescent with gene transfer C A pin copper wire carbon adhesive cyanacrylate y y adhesive glass carbon fiber silicone elastomer small clear vesicles neuronal dense core vesicles ‘chromaffin granule-like’ large vesicles 28 Two different types of brain cells made to glow green and red using gene transfer Green Fluorescent Protein Red Fluorescent Protein Getting the Gene out of the Cell Mature RNA DNA made by “reverse transcription” Reverse transcriptase allows generation of the DNA version of the mature RNA The moral: do not believe dogmas! 29 The basic toolkit for DNA manipulation: 1. DNA which encodes the gene (must be sequenced for further analysis) 2. Restriction endonucleases (special enzymes) to cut and other enzymes to glue bits of DNA together g g 3. Plasmids to proliferate DNA 4. PCR (to generate numerous copies of the sequences we know, to introduce mutations and “sticky ends”) Restriction Endonucleases DNA sequence etc … approx. 5000 bp 30 Make a map with restriction sites EcoR1 EcoR1 Plasmids: Small self-replicating DNA molecules which live in bacteria 31 DNA of interest EcoR1 5’ G A T T C 5’ Plasmid DNA EcoR1 C T A A G EcoR1 C T A A G EcoR1 G AT T C C T A A G “Sticky ends” find each other! DNA ligase G AT T C C T A A G MOVIE PLASMID CLONING 32 Putting the Gene Back into the Cell Our gene delivery vehicles Adenoviral vectors E1 Early ‘E’ genes 0 ITR E3 E4 E2 20 40 L1 L2 L3 60 80 L4 100 36 kb genome ITR Late ‘L’ genes L5 Late transcription TRANSGENE 33 WHAT IS IT WE NEED TO DO TO “EXPRESS A GENE?” PROMOTER THE GENE POLY-A signal 3’ end 5’ end An example of an expression cassette m RNA TRANSCRIPTION FACTOR(S) How can we make these vectors selective for certain types of cells? Promoter sequences 5’ Transcription factors RNA polymeraseII Coding part of the gene • Transcription factors bind to SPECIFIC SEQUENCES in gene promoters and facilitate transcription… Each cell is producing its individual set of proteins because… each cell has a specific blend of active transcription factors A B THE GENE POLY-A tail 3’ end 5’ end PROMOTER Binding sites for the TRANSCRIPTION FACTOR(S) m RNA 34 Two different types of brain cells made to glow green and red using gene transfer Gene manipulation in the brain reduces blood pressure in an animal model of hypertension (spontaneously hypertensive rat) EGFP in SHR 160 Systolic Blood 140 pressure p<0.05 120 100 eNOSi in SHR eNOSi in WKY 0 7 14 Waki et al Hypertension 2006 Summary 1. DNA fragments may be cut out of their original context and re-cloned into new DNA molecules such as plasmids. 2. Foreign genes may be expressed in various cells. An expression cassette may be placed into a viral vector to deliver this gene to differentiated cells in the body. body 3. It is possible to target genes to specific cell types in the body (selected brain cells, cardiac muscle cells, blood cells etc) using cell-specific promoter sequences. 4. Viruses may be genetically modified and used as gene delivery tools. This is one of the approaches to human gene therapy. 35 Can you suggest a caption for this picture? SEE YOU LATER, ALLIGATOR!!! 36