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Seminars in Bio200 • The UW has hundreds of excellent public talks from top scientists every quarter, but most students do not access this unique resources until they are in their final year (if at all). • To help you step into the world of cutting edge science, we are giving 30 points for completing this Seminar assignment. You’ll need to: – Find a seminar that is likely to contain some Bio200 material (anything with cells, genes, biotechnology, biomolecules, etc) – Go! • You won’t understand everything, and that is OK! • Try to relax and enjoy the experience – Afterwards, complete the Seminar Survey • You might want to take a look at this beforehand so you know what you’ll be answering. – Due: Before the start of the last class of the quarter. January 17th, 2016 Class 9 Learning Goals Information Flow in the Central Dogma Model • After this class, you should be able to: – In broad terms, identify the movement of molecular information – Appreciate the historical significance in finding the molecular unit of genetic information – Within the ‘Central Dogma’ model of genotype to phenotype: – Define and describe ‘transcription’ – Define and describe ‘translation’ – Identify potential phenotype-changing mutations in DNA Peer Instruction What are some examples of things that contain information? How do you know? Is information a physical thing? Define ‘information’: Peer Instruction Coiled 1) What is supercoiling? Supercoiled DNA • One base pair = 0.34 nm (3.4E-9 meters) • Base pairs in the genome of each of your cells: ~3 billion 2) Why is supercoiling important? Supercoiled DNA in chromosome 1. Define transcription: 2. Define translation: 3. Is transcription without translation useful in any way? 4. Is translation without transcription useful in any way? 5. Are all protein-coding genes read by the same cellular machinery? 6. The Central Dogma of Molecular Biology describes the most common path of information flowing from a source into the design of working molecules. Draw a simple diagram of the CDoMB: Peer Instruction Peer Instruction 3ʹ 5ʹ 5ʹ 3ʹ 5ʹ 3ʹ Nʹ Cʹ Nʹ Cʹ 5ʹ 3ʹ Between these two alleles of the same gene: • Has the genotype changed? • Has the phenotype changed? AGU CAU CAGACC GAAU CCGU ACG UU AGG CAU G C GUA GUC UG OH • • • • • • • COO- HN Concept Questions S Why is DNA more likely to be the information storage molecule than RNA? Why is protein more likely to be the working molecule than RNA? Name two different ways that molecular information stored in the central dogma? Without gaps, describe the progression of information from DNA to protein. Define and describe ‘transcription’ Define and describe ‘translation’ In the given worksheets, identify and explain a potential phenotype-changing mutation in DNA. – How is this mutation changing information? – Why is this mutation not guaranteed to change phenotype? Peer Instruction Proteins that should go to the Rough ER must be identified. How does this information become encoded in the protein? Cytosol Ribosome SRP RNA Signal sequence Emerging Protein Lumen of rough ER SRP receptor Protein Wednesday, January 18th, 2017 Class 10 Learning Goals Transcription • After this class, you should be able to: – Label each molecule and strand (and give correct polarity for each nucleic acid) in a diagram of a particular transcription – Predict and give a rationale for the effect of a loss-of-function mutation in any component of prokaryotic transcription – Compare or contrast the three phases of prokaryotic trancription (initiation, elongation, termination) in terms of enzymes and substrates – Given a DNA sequence and promoter, predict the: – Direction of transcription – Polarity and type of the new molecule – Sequence of the new molecule Transcription Non-template (coding) strand Peer Instruction DNA RNA 5ʹ 5ʹ 3ʹ 3ʹ Phosphodiester bond is formed by RNA polymerase after base pairing occurs 5ʹ 3ʹ 3ʹ 5ʹ Template strand RNA 5ʹ 3ʹ Hydrogen bonds form between complementary base pairs DNA template 3ʹ What is “5’ to 3’ polarity”? What is happening in this diagram? 5ʹ 1) Label the 5->3 polarity of every strand. Peer Instruction 2) Label the ‘template’ and ‘non-template’ strands 3) Label the ‘upstream’ and ‘downstream’ directions. Peer Instruction • Label the +1 site • Where are – (minus) sequences numbered? Peer Instruction Label: DNA molecule Two different proteins Label any: Protein/DNA binding zones Protein/protein binding zones Peer Instruction Label: • A region of DNA called the ‘promoter’ • The enzyme called ‘RNA polymerase’ • A small protein called ‘sigma’ • What is a promoter? • • What does sigma do? Peer Instruction Label the -10 box and -35 box and explain their names. Label the upstream and downstream regions. +1 • • • What has changed? Label the +1 site What are the yellow molecules? Peer Instruction Peer Instruction • What changed from the previous diagram? Rudder Zipper • What do the “zipper” and “rudder” do? Peer Instruction • • What is the transcription termination signal? What is the yellow structure on the lower left? Peer Instruction Enzyme binding to the termination hairpin Two methods of terminating transcription ‘Intrinsic’ disruption by the new RNA itself active site • The Transcription Problem: Concept Questions – Create a stretch of double stranded DNA with random sequence – Flip a coin, and place the promoter to the left if heads (right if tails) and assume that the promoter points the RNA polymerase towards the sequence in either case. – Flip the coin again, and put 3’ on the top right end of the strand if heads (5’ if tails). – Now: What is the new RNA molecule sequence and polarity? • What would happen to transcription if the -10 and -35 boxes were switched? What if the +1 was a different base? What if the termination sequence was lost? • There are four channels in the RNA polymerase protein leading from the core to the outside. Name each of these channels usefully based on their functions • Compare and contrast the three phases of prokaryotic trancription (initiation, elongation, termination) in terms of enzymes and substrates • Transcription initiation starts right at the promoter. Why does termination of transcription not occur until many bases after the termination signal? Thursday January 19st, 2017 Class 11 Learning Goals Translation • After this class, you should be able to: – Label each molecule and strand (and give correct polarity for each nucleic acid and amino acid polymer) in a diagram of protein translation – Predict and give a rationale for the effect of a loss-offunction mutation in any component of the ribosome – Compare or contrast the three phases of translation (initiation, elongation, termination) in terms of enzymes and substrates Peer Instruction An ‘adapter molecule’ is needed to hold amino acids and interact with mRNA codons. Amino acids Adapter molecules mRNA Codon Codon Codon Codon Why didn’t transcription require an adapter molecule? How many total adapter molecules are needed? Because NTPs and dNTPs can directly forms base-pairs Peer Instruction 1) What are the important parts of a Transfer RNA (tRNA)? Binding site for the amino acid 3ʹ 5ʹ 3ʹ 5ʹ 3ʹ mRNA 5ʹ 5ʹ mRNA 3ʹ 2) How is a tRNA “charged”? The machine that does translation: The Ribosome tRNA in A site (red) Ribbon model of ribosome during translation tRNA in E site (blue) tRNA in P site (green) Large subunit Small subunit Ribosomes catalyze peptide bond formation Peer Instruction How does this enzyme create a charged tRNA? ATP 1) 3) 4) 2) Activated enzyme complex What parts of the enzyme give it specificity? Concept questions • To practice labeling of translation diagrams: – Build a sequence with 70+ bases of random RNA – Find a start codon and assess the location of ribosome binding – Indicate the codons and anticodons used, as well as the peptide bonds created – Change the sequence of the RNA such that you program the ribosome to create a 10-amino-acid polymer • What would happen to translation if: – The ribosome binding site were lost? – The start codon were mutated? – The stop codon was mutated – The shape of the release factor was altered • Write a complete reaction diagram for each of these: – Binding of the small ribosomal subunit to the RNA – Addition of the 5th amino acid – Termination of translation – Catalysis of charging a single tRNA Lab Next Week • The Central Dogma Lab – A great place to practice what you know about • Trancription • Translation • Mutation – We’ll also talk about mutations and their impact on human disease for the first time