How many processes can you name?
The Big Picture (Shown at very beginning of class, before everyone is seated) If you would like to see a narrated version, its here: How many processes can you name? Lecture 0 Basics of Molecular Biology
Welcome to lecture 0! SynBUM | MIT iGEM Team 2010 Create Your Own Bacterial Air Freshener 1/6/2011 What you will learn in this lecture
The cell as the basic unit of life Structure of Important Macromolecules DNA RNA Proteins The Central Dogma Transcription Translation Regulation Correlation to Synthetic Biology Cells - Fundamental working units of every living system.
Organelles Nucleus (contains DNA) Cytoplasm Membrane DNA (no nucleus) Eukaryotic cell Prokaryotic cell Every organism is composed of one of two types of cells: prokaryotic cells or eukaryotic cells. A eukaryotic cell has membrane-enclosed organelles, the largest of which is usually the nucleus By comparison, a prokaryotic cell is simpler and usually smaller, and does not contain a nucleus or other membrane-enclosed organelles Bacteria and Archaea are prokaryotic; plants, animals, fungi, and all other forms of life are eukaryotic Prokaryotes andEukaryotes are descended from the same primitive cell all life on Earth is the result of 3.5 billion years of evolution. The cell is the lowest level of organization that can perform all activities required for life All cells: Are enclosed by a membrane Use DNA as their genetic information The ability of cells to divide is the basis of all reproduction, growth, and repair of multicellular organisms Comparison of Prokaryotic and Eukaryotic Cells
Prokaryotes Eukaryotes Single cell Single or multi cell E. coli chromosome: 4X106 bp Yeast chromosome: 1.35x107 bp 90% of DNA encode protein Small fraction of DNA encodes protein: Many repeats of non-coding sequences No nucleus Nucleus No organelles Organelles One piece of circular DNA Chromosomes No mRNA post transcriptional modification Exons/Introns splicing Prokaryotic cells are generally chosen for genetic engineering because: Processes are better understood to engineer something intelligently we need to understand what were changing first! Easier to maintain, less specialized equipment needed Faster, easier to scale up However, as can be seen in the last iGEM project (well talk about this later), Eukaryotic cells also have a wide range of outputs. In our class well be using prokaryotic cells All Cells Divide All cells go through similar cycles: they eat, grow, replicate their DNA, divide, and repeat The microraphs have the cytoskeleton stained, showing cell division: Above: early anaphase Below: telophase Cell Cycle: The Chromosomal View
(b/c its pretty ) The Central Dogma DNA RNA Proteins
Control/ Info Center -Genes -Regulatory elements The messenger -mRNA, tRNA, rRNA Also can be: -Ribozymes -siRNA The machinery -Enzymes -Signaling -Replication -Many more DNA Replication The central dogma is just what it sounds like: it is central to our current understanding of biology. Information encoded in DNA is passed to RNA and expressed as proteins. Control/Info Center: (DNA & RNA): Stored as nucleic acids: biological molecules(DNA and RNA) Specific sequences of DNA bases that encode instructions on how to make proteins. Machinery/Factory + Products (Proteins): Building blocks of cells Collect and manufacture components, form enzymes Carry out replication Genotype: The genetic makeup of an organism (whats inside) Phenotype: the physical expressed traits of an organism (what you see) Of course, in recent years weve found that the cell has many levels of regulation beyond the simple DNA->RNA->Protein path. However, for this lecture we will focus on the central dogma. DNA Transcription RNA Translation Proteins Information encoded in DNA is passed to mRNA Information carried by mRNA is used to make proteins But first, what are DNA, RNA, and Proteins? Where are we? The cell as the basic unit of life
Structure of Important Macromolecules DNA RNA Proteins The Central Dogma Transcription Translation Regulation Correlation to Synthetic Biology DNA: The Code of Life 5' end 5'C 3'C Nucleoside Nitrogenous base
(b) Nucleotide Nucleoside Nitrogenous base Phosphate group Sugar (pentose) DNA: Deoxyribonucleic acid Polymer nucleotides Purines (these are bigger): Adenine and Guanine Pyrimidiens (Smaller): Thymine and Cytosine The two strands are held together by H-bonds A pairs with T, C pairs with G Antiparallelhas a 3 (phosphate) end and a 5 (hydroxyl) end DNA encodes many things: Genes that tell the cell how to make proteins As well as tandem repeats, trash, regulatory elements, protein binding sites, etc Theres still a lot we dont know about DNA! Nucleoside triphosphate
DNA Replication A C T G New strand 5 end Template strand3 end 5 end 3 end Nucleoside triphosphate Pyrophosphate DNA polymerase Semiconservative Template DNA is used by DNA polymerase to synthesize the new strand Proceeds only from the 5 to 3 end (Aside) For the chemists, why this this happen? Answer: polymerization requires a free hydroxyl group DNA Replication (E. Coli)
Leading strand Lagging strand Origin of replication Primer Overall directionsof replication Origin of replication Template Strand New Strand dsDNA (a) Origins of replication in E. coli 0.5 m DNA Replication (Eukaryote)
0.25 m Origin of replication Double-stranded DNA molecule Template Strand New Strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes RNA RNA DNA Single Stranded Double Stranded Temporary (mRNA) Stable
Uracil Thymine Ribose Deoxyribose mRNA tRNA rRNA miRNA/siRNA Ribozymes Main differences between RNA and DNA. There are many types of RNA known Shown are two view of tRNA tRNA linear and 3D view: Main types of RNA mRNA this is what is usually being referred to when we say RNA.This is used to carry a genes message after transcription tRNA transfers genetic information from mRNA to an amino acid sequence during translation rRNA ribosomal RNA.Part of the ribosome which is involved in translation Proteins are made of Amino Acids
group Carboxyl carbon Proteins are a chain of 20 different amino acids different chemical properties cause the protein chains to fold up into specific three-dimensional structures that define their particular functions in the cell. 3 letters of RNA(/DNA), called a codon 1 amino acid 64 possible combinations map to 20 amino acids Degeneracy of the genetic code- several codons to same amino acid Proteins do all essential work for the cell build cellular structures digest nutrients execute metabolic functions Mediate information flow within a cell and among cellular communities. Proteins work together with other proteins or nucleic acids as"molecular machines" structures that fit together and function in highly specific, lock-and-key ways. Proteins are the Workhorses of the Cell
Primary Structure Secondary Structure Tertiary Structure Quaternary Structure pleated sheet +H3N Amino end Examples of amino acid subunits helix A ribbon model of lysozyme (a) (b) A space-filling model of lysozyme Groove Primary structure: amino acid sequence, determined by DNA Secondary structure: Hbonding between backbone Tertiary structure: Hbonding, Hydrophobic forces, disulfide bonds, etc. between R groups Quaternary structure: how many polypeptides fit together The shape of a protein is extremely important for its function! Little changes in shape can lead to nonfunctional proteins. Where are we? The cell as the basic unit of life
Structure of Important Macromolecules DNA RNA Proteins The Central Dogma Transcription Translation Regulation Correlation to Synthetic Biology The Central Dogma Revisited
DNA molecule Gene 1 Gene 2 Gene 3 template strand TRANSCRIPTION TRANSLATION mRNA Protein Codon Amino acid Slight Variations of the Central Dogma
(b) Eukaryotic cell TRANSCRIPTION Nuclear envelope DNA Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide TRANSCRIPTION DNA mRNA TRANSLATION Ribosome Polypeptide a) Bacterial Cell Transcription Terminology
Phosphodiester Bond Promoter RNA (ribonucleotide) RNA Polymerase II Terminator Phosphodiester Bond: Esterification linkage between a phosphate group and two alcohol groups. Promoter: A special sequenceof nucleotides indicating the starting point for RNA synthesis. RNA (ribonucleotide): Nucleotides A,U,G, and C with ribose RNA Polymerase: Multi-subunit enzyme that catalyzes the synthesis of an RNA molecule on a DNA template from nucleoside tri-phosphate precursors. Terminator: Signal in DNA that halts transcription. Transcription Initiation Elongation Termination
Promoter Transcription unit DNA Start point RNA polymerase 5 3 Initiation 1 RNA transcript Unwound Template strand of DNA 2 Elongation Rewound 3 Termination Completed RNA transcript Transcription 3 Main stages: Initiation, elongation, and termination Catalyzed by RNA Polymerase Eukaryotes process mRNA; this does not occur in prokaryotes. Transcription occurs in the nucleus. Initiation Promoters signal the initiation of RNA synthesis Transcription factors mediate the binding of RNA polymerase and the initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Elongation As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases RNA polymerase II catalyzes the formation of phosphodiester bond that link nucleotides together to form a linear chain from 5 to 3 by unwinding the helix just ahead of the active site for polymerization of complementary base pairs. The hydrolysis of high energy bonds of the substrates (nucleoside triphosphates ATP, CTP, GTP, and UTP) provides energy to drive the reaction. During transcription, the DNA helix reforms as RNA forms. Termination The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA Translation Terminology
Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly Codons 3 5 mRNA Translation Terminology Codon mRNA Ribosome rRNA tRNA Anti-codon C-Terminal N-terminal Codon: The sequence of 3 nucleotides in DNA/RNA that encodes for a specific amino acid. mRNA (messenger RNA): A ribonucleic acid whose sequence is complementary to that of a protein-coding gene in DNA. Ribosome: The organelle that synthesizes polypeptides under the direction of mRNA rRNA (ribosomal RNA): The RNA molecules that constitute the bulk of the ribosome and provides structural scaffolding for the ribosome and catalyzes peptide bond formation. tRNA (transfer RNA): The small L-shaped RNAs that deliver specific amino acids to ribosomes according to the sequence of a bound mRNA. Anticodon: The sequence of 3 nucleotides in tRNA that recognizes an mRNA codon through complementary base pairing. C-terminal: The end of the protein with the free COOH. N-terminal: The end of the protein with the free NH3. Translation Accuracy Accurate translation requires two steps:
P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) mRNA binding site Large subunit Small Next amino acid to be added to polypeptide chain Amino end Growing polypeptide tRNA E P A Codons 5 3 Requires 2 correct matches: Between tRNA and correct amino acid Between tRNA codon and mRNA anticodon Accurate translation requires two steps: First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase Second: a correct match between the tRNA anticodon and an mRNA codon Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) Building a Polypeptide
The three stages of translation: Initiation Elongation Termination All three stages require protein factors that aid in the translation process RNA to Protein: Instruction Book of Life
Start with Methionine End with a stop codon Note the degeneracies for each amino acid Translational Initiation
Large ribosomal subunit 3 U 5 A C P site Met 5 A Met U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit that completes the translation initiation complex Small ribosomal subunit mRNA binding site Translation initiation complex Translational Elongation
Amino end of polypeptide mRNA 5 3 E P site A GTP GDP Ribosome ready for next aminoacyl tRNA During the elongation stage, amino acids are added one by one to the preceding amino acid Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation Translational Termination
Release factor 3 5 Stop codon (UAG, UAA, or UGA) 2 Free polypeptide 2 GDP GTP Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid This reaction releases the polypeptide, and the translation assembly then comes apart Almost-Unsimplified Overview
Based on crystallographic data, real protein structures The only simplifications are: time scale, less crowded background Where are we? The cell as the basic unit of life
Structure of Important Macromolecules DNA RNA Proteins The Central Dogma Transcription Translation Regulation Correlation to Synthetic Biology The cell is not a sac of chemicals
Homeostasis Levels of Gene Regulation Pre-transcriptional Pre-translational Post-translational Gene/protein interactions Negative Feedback Positive Feedback A cell is much more than just a sac of chemicals. It is highly regulated and ordered. The process of homeostasis maintains a balance between processes and conditions inside and outside the cell Regulation of a gene can occur at 3 different levels Pre transcriptional: this is the most energy efficient level. For example, transcription factors stimulate or prevent transcription of a certain gene. There are also other ways to regulate the gene, such as chromatin condensation through histone modifications Pre-translational: this can be: alternate splicing of a gene Increased degredation/ stability of mRNA mRNA interference Increase degredation Prevent docking onto ribosome Post-translational: most energy cost, after protein has already been made Increased degredation Protein cleavage: can activate protein Phosphorylation Feedback mechanisms allow biological processes to self-regulate Negative feedback means that as more of a product accumulates, the process that creates it slows and less of the product is produced Positive feedback means that as more of a product accumulates, the process that creates it speeds up and more of the product is produced Negative Feedback A Negative feedback Enzyme 1 B D Enzyme 2 Excess D
blocks a step Negative feedback D C B A Enzyme 1 Enzyme 2 Enzyme 3 Positive Feedback W Enzyme 4 X Positive feedback + Enzyme 5 Excess Z Y
stimulates a step Z Positive feedback Enzyme 4 Enzyme 5 Enzyme 6 Y X W + Protein Interactions within the cell
Theres still a lot more we dont know!! Synthetic Biology: How to get the cell to do what we want?
Mix and match promoters, regulatory sites, and coding sequences to build logic circuits Does this look familiar to anyone? This is the circuit the 2010 iGEM team built in bacteria. UV lighttogglephage polymerization + fluorescence. The possibilities are endless!
Control signaling pathways Search and destroy: cancer cells, pathogens Control metabolic pathways Clean up oil spills Make novel biomaterials Hijack the cells differentiation pathways to direct differentiation T-cell differentiation cure AIDS Make artificial organs Make new pathways Electricity generating bacteria Can you think of a few? Visit previous iGEM websites to see what other college students have done!