Cell Biology with Todd Hennessey Week Thirteen Notes
Cell Biology with Todd Hennessey Week Thirteen Notes BIO 201
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This 37 page Class Notes was uploaded by ChiWai Fan on Saturday April 30, 2016. The Class Notes belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 103 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
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Date Created: 04/30/16
Cell Bio on April 25, 27, 29, 2016 (All images taken from Professor Hennessey’s slide—edited by ChiWai Fan) Summary of Results: 1. Griffith: A heat-stable transformation factor, present in virulent strains of pneumococcus, can transform a non-virulent strain into killers. What is it? He didn’t know. It is a genetic transformation though. 2. Avery: The transformation factor is destroyed by DNAse so it is DNA. 3. Hershey-Chase: Metabolic labeling of phage (viral) DNA showed that DNA is the genetic material passed from parent to progeny. True or False? DNA is always the genetic material passed from parent to progeny. FALSE. We know there are RNA viruses that has only RNA inside, no DNA. But they didn’t know this back then. RNA VIRUS: No DNA in this type of virus. Only RNA Progeny have RNA and no DNA An RNA virus contains RNA as its genetic material. It can be single-stranded RNA (ssRNA) but may be double-stranded RNA (dsRNA). Notable human diseases caused by RNA viruses include Ebola, hemorrhoragic fever, SARS, influenza, hepatitis C, West Nile fever, polio, and measles. Viruses are not cells; viruses are not alive So, DNA is the genetic material passed from parent to progeny IN CELLS. DNA Replication: we focus on eukaryotic Two main steps: 1. The two strands of the double helix are unwound and opened up 2. Complementary base pairs (C-G, T-A) line up and get joined together to form the new DNA copy Four key features of eukaryotic DNA DNA is a double-stranded helix with complimentary base pairs (C-G,T-A) DNA is usually a right-handed helix DNA strands are antiparallel (they run in opposite directions) DNA has major and minor grooves DNA Is a Double Helix The major groove is wider than the minor groove Hydrogen bonding between GC and AT pairs causes the amount of A to be equal to T and amount of G equal to C DNA Grows from 5’ to 3’: DNA polymerase adds deoxyribonucleotide (dNTPs) to the 3’ end 1. Nucleotides (dNTPs) are added to the 3’ end always 2. The enzyme DNA polymerase adds the next deoxyribonucleotide to the –OH group at the 3’ end of the growing strand and releases pyrophosphate 3. Bonds linking the phosphate groups are broken, releasing energy to drive the reaction The Origin of DNA Replication: think of a bubble 1. The ori sequence binds the pre-replication complex 2. Two replication folks move away from one another 3. There are multiple origins of replication for eukaryotes to get things done faster 4. Replication folks move away from each other DNA Replication “DNA polymerase is used to replicate DNA when the cell is undergoing mitosis or meiosis.” True or False? FALSE. No DNA replication in MITOSIS & MEIOSIS. DNA replication is in S PHASE!! Some requirements for DNA replication It must be semi-conservative. o DNA replication is the process of producing two identical replicas from one original DNA molecule o It only happens during S-phase. It must proceed 5’ to 3’ o Deoxyribonucleotide triphosphates (dNTPs) are added to the 3’ end in DNA replication It requires a template o For DNA replication, parental DNA is the template It requires a primer o For most DNA replication, the primer is RNA. Can’t start DNA replication without a short piece of RNA The energy for replication comes mostly from either ATP or dNTPs The cast of characters for DNA replication 1. Helicase—takes apart or unwinds the helix of DNA 2. DNA Polymerase III 3. Sliding Clamp—helps keep polymerase on the DNA 4. Single-Strand DNA binding Protein (SSB)—occupying any open nucleotides so they don’t bind to random things 5. DNA Primase—it is an RNA polymerase that makes the primer 6. DNA Polymerase I—it makes DNA 7. Ligase—to join together in; seals gaps of DNA DNA replication animation 1. Helicase binds and unwind the DNA helix 2. DNA polymerase, with the clamp, bind to the leading strand (leading strand synthesis is continuous; legging strand discontinuous) 3. Single-stranded DNA binding proteins (SSB) bind to exposed strand; prevent re-annealing of the DNA strands 4. Leading strand synthesis proceeds 5’ to 3’ continously; always adding to the 3’; DNA Polymerase III synthesizes DNA in a 5’3’ direction. A sliding clamp tethers the DNA pol III to the template making the polymerase processive 5. Lagging strand synthesis: Okazaki fragments are made on the lagging strand; discontinuous 6. DNA synthesis starts with an RNA primer; DNA primase synthesizes a short RNA primer; then it comes off, only to return later to start the next fragment DNA polymerase extends the fragment from 5’ to 3’ In actuality replication does not take place in discrete steps. The replication machinery allows all these steps to take place in concert 7. Ligase joining the fragments together 8. The process continues 9. As the fork extends, the process repeats, forming a continuous leading strand and multiple Okazaki fragments FROM THE BOOK: Many Proteins Collaborate in the Replication Complex DNA polymerase elongates both strands 1. DNA helicase unwinds the double helix 2. Single-strand binding proteins keep the template strands separated 3. Primase synthesizes a primer 4. DNA polymerase elongates both strands The book didn’t include DNA clamp, ligase, and telomerase. We want to make sure there’s still telomers here, telomerase goes in to make it longer. DNA synthesis starts with a primer Primase synthesizes an RNA primer Primase makes an RNA copy DNA polymerase replaces the primase so that a new strand of DNA can be made DNA polymerase makes a DNA copy The Two New Strands Form in Different Ways Remember: DNA synthesis must go from 5’ to 3’ Leading strand: Continuous replication Lagging strand: Okazaki fragments and discontinuous; have to wait for it to open up more to make another fragment; when you made Okazaki fragments, we have to remove RNA primer, and fill in gap with ligase The gaps between the Okazaki fragments are joined by DNA ligase Polymerase comes off, ligase goes on A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization This makes the polymerase processive instead of coming off after 50-100 nucleotides. Sliding DNA Clamp keeps them on so it increases efficiency 1. A clamp binds to the DNA 2. DNA polymerase binds to the clamp-DNA complex 3. The clamp keeps the polymerase stably bound to DNA so that many nucleotides can be added for each binding event Another view from another book: bacterial 1. Enzyme unwind the parental double helix 2. Protein stabilize the unwound parental DNA 3. The leading strand is synthesized continuously by DNA polymerase 4. The lagging strand is synthesized discontinuously, RNA polymerase synthesiszed a short RNA primer, which is then extended by DNA polymerase 5. DNA polymerase digests RNA primer and replaces it with DNA 6. DNA ligase joins the discontinuous fragments of the lagging strand DNA polymerase can proofread DNA during replication, but it is not perfect 1. During DNA replication, an incorrect nucleotide may be added to the growing chain 2. The proteins o the replication complex immediately excise the incorrect nucleotides 3. DNA polymerase adds the correct nucleotide and replication proceeds Correcting errors after replication cut it out and edit it 4/27/16 “One gene, one enzyme”? Archibald Garrod in the early 1900s His hypothesis: A gene defect an enzyme causing it to do something Inborn errors of metabolism: mutant allele produces inactive enzyme and it accumulates which causes black urine. The one gene-one enzyme hypothesis is the idea that genes act through the production of enzymes, with each gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway. We now know that the hypothesis is wrong. How to test this hypothesis (one gene, one enzyme) Making mutations in the lab What is the function of an identified gene? Two basic ways Forward genetics. From phenotype to genotype Random mutagenesis Screen or select for mutants (phenotype: growth) Use a phenotype to identify the altered gene. Forward genetics is looking at a phenotype and try to use ha t to find the gene responsible for that phenotype Reverse genetics. From gene to function (phenotype) remove something to see if it is the cause of the function Start with a known gene Disrupt the gene (by mutation, knockout, etc.) and see what effects it has on the phenotype Using forward genetics to test the “one gene, one enzyme” hypothesis Beadle and Tatum (1941) Random mutagenesis produces many unknown mutations in a bread mold Use wild type and mutagenized them Isolate clones and grow up many clonal lines so they’re all the same See if any die in minimal media. Those may be mutants that are lacking an enzyme activity Beadle and Tatum continued They added different vitamins and amino acids until they found something that a mutant can grow on Add Arginine and they grew. This suggested that the mutants couldn’t make arginine. Wild type (normal) can make it, even in minimal medium Looking for the clonal line that only has one mutant in one enzyme and find the gene that’s responsible for this enzyme Srb and Horowitz followed this up in 1944 They found more mutants and used them to put together a biochemical pathway Strains isolated and added stuff: 1. Strain 1 needs arginine to grow. It can’t grow on citruline. It has a defect in arginine synthesis (we will find out that citruline is necessary to make arginine) 2. Strain 2 can grow on either arginine or citruline but not on ornithine. It has a defect in citruline synthesis 3. Strain 3 has a defect in ornithine synthesis Why do this? What did they want to know? That means they lack enzyme to make this thing and that there’s gene mutation in that gene for the enzyme. This is Forward Genetics approach. Identifying a biochemical pathway What genes are involved? At least four genes responsible to make Arginine (four chances for gene mutation) How about a Reverse Genetics approach? Question: Is Gene B a citruline synthetase? Hypothesis: Gene B codes for the enzyme citruline synthetase. A Gene knockout (remove the gene) of gene B will produce a mutant that cannot grow on ornithine Experiment: Remove gene B from the cell. This will produce a gene B knockout cell line. Test the mutants to see if they can grow with and without citruline Prediction: If the knockout can only grow if citruline is added, it suggests that gene B is a citruline synthetase Follow-up experiment: Assay the production of citruline from ornithine in mutant and wild type. An example of a gene knockout (Gene replacement) 1. Identify and clone the gene 2. Using current techniques, construct a piece of DNA that has the gene replaced with a drug resistance marker 3. Transform the cell of interest with this modified DNA 4. The disrupted gene can integrate into the genomic DNA at a very low frequency, depending on the cell 5. How can you tell if the cell has this gene disruption? It will be resistant to the drug 6. Other techniques can be used to verify that the gene product is not produced. Can you think of one from BIO201B? Does this cell still produce the protein? Gene A produces mRNA to make the protein. Can’t make protein without Gene A. Run on SDS-PAGE to see if there’s a missing band. How to find out if a gene is really knockout: SDS-PAGE Exceptions we now know to “one gene, one enzyme” 1. Not all proteins are enzymes; enzymes are catalysts. A. Some gene products (proteins) are structural (like tubulin, actin, etc). B. Some are co-factors or regulators (like cyclin) Beadle and Tatum noted that it was "entirely tenable to suppose that these genes which are themselves a part of the system, control or regulate specific reactions in the system either by acting directly as enzymes or by determining the specificities of enzymes“. 2. Not all enzymes are made from only one gene Some are multiple protein enzyme complexes (Quaternary structure) 3. Some genes can make multiple proteins (like pre-pro hormones) 4. Exon shuffling can change protein expression Protein hormone processing One gene can make many proteins This gene makes things that make other things. This gene makes 8 different proteins. From Gene to Protein Transcriptionprocessingtranslation. (From gene to protein) Old “Central dogma”—this is wrong Current model: Reverse transcription Transcription—making RNA from DNA template A. Initiation—open and unwind DNA; This RNA polymerase makes RNA from DNA template B. Elongation— nTPs are added to the 3’ end for elongation C. Termination—something about the sequence that tells it when to stop; this only makes one piece of RNA. Only one strand of the DNA acts as a template Initiation of transcription: This needs: 1. Opened and unwound DNA. This RNA polymerase has helicase activity. Bind RNA polymerase to promoter 2. Promoter region on the DNA template. The promoter is not transcribed; not all genes are transcipted) Ex. P53 binds to the promoter of p21 3. RNA polymerase to make RNA 5’ to 3’ on the template strand; (add polymerase to 3’ growing end; Elongation: Transcription produces an RNA transcript. It is complementary to the DNA template strand except: o It has ribonucleotides instead of deoxyribonucleotides (dNTPs) o It has U instead of T After termination, the RNA polymerase complex comes off to be used again The full RNA transcript is now seen Two other views of transcription Since nucleotides are added to the growing 3’ end, the 5’ end comes out first Regulation of gene transcription Potential Points for the Regulation of Gene Expression “From gene to protein” Chromatin remodeling Regulation of transcription RNA processing RNA export from the nucleus RNA degredation Regulation of translation Post-translational modifications and protein degredation 4/29/16 Chromatin remodeling Chromatin remodeling helps to unwrap nucleosomes Genes cannot be transcribed when they are wrapped around histones as nucleosomes. The DNA has to come off of the histones to be transcribed; regulate gene expression by how tight histones are wrapped around it or rewind the histones. The DNA has to come off the histone to get transcribed. Nucleosome: Acetylation takes away positive charges on histones: Takes away charge, now DNA is no longer attached to it and now it can be expressed Histone acetyltransferase (HAT) Regulation of gene transcription There are many genes that are not expressed until they are told to do it Can you tell me one example from BIO201B so far? P53 Positive and Negative Regulation Binding of a repressor protein blocks transcription—turns off this gene Binding of activator protein stimulates transcription (reducer)—turns on this gene An Inducer Can Stimulate the Expression of a Gene for an Enzyme Note that after the inducer is removed, the enzyme activity remains even after the level of mRNA transcription of this gene goes down. Why? Can’t always accurately predict the amount of protein just by knowing the expression of the mRNA. What makes the level of mRNA go down? RNAses; it breaks down RNA. (mRNA synthesis vs. mRNA breakdown) Add inducer, level of B-Galactosidase goes up; Remove inducer, mRNA go down after a while but the protein is still there. The point? Every cell in your body that contains DNA has all of the genes to do everything A skin cell has the genes to be a heart cell. They’re just not expressed Cell differences is due to gene expression There are some genes in some cells that are never transcribed Many genes are silent until induced. Most genes are regulated at the level of transcription. Transcription factors (and other things) can tell a gene to either make a transcript or not. If transcription factor is there, then you make the gene. (inducers can control things in cell cycle.) True or False? All of our DNA sequences contain genetic information that code for proteins. False. Some parts of DNA are never transcribed but are replicated: 1. Telomeres 2. Centromeres These DNA sequences do not code for proteins What other DNA sequences do we know of that do not code for proteins? 1. The DNA sequences that code for rRNA --Majority of the RNA you produce in a cell is never translated. 2. The DNA sequences that code for tRNA 3. Others (like intron sequences, promoters, etc.) Promoter is a signal to tell a gene to be transcipted but it is not transcipted itself. True or false? All DNA is transcribed into RNA--FALSE; All RNA is translated into proteins--FALSE Introns and exons Only the Exon sequences are transcribed to make this particular mRNA. Some genes do not have introns. Introns are cut out. The time when you can have RNA as an enzyme to do splicing. Not all enzymes are proteins. Other highpoints: 1. Some DNA is never transcribed (Ex: telomeres, centromeres) 2. Of the DNA which is transcribed, not all of it codes for proteins (Ex: tRNA, rRNA, introns) 3. A gene that is transcribed in one cell type might not be expressed in another: G1 cyclin shouldn’t be expressed in a G2 cell. Synaptonemal complex genes should only be expressed in Prophase I of Meiosis I. (Ex: G1 cyclin, synaptonemal complex genes, etc.) 4. “One gene, one protein” is no longer true (Ex: Alternative splicing, protein processing, etc.) SUMMARY mRNA processing: eukaryotic mRNA Splicing This happens in the nucleus; Introns are transcribed but not translated; because theyre removed before translation. The mRNA can be cut, remodeled and modified before it is translated. There are many other possible modifications that are not shown here 1. The exons and introns of the coding region are transcribed 2. The introns are removed 3. The spliced exons are ready for translation after processing Alternative Splicing One gene, one enzyme? One gene can make several different proteins: exon shuffling Processing the Ends of Eukaryotic Pre-mRNA When mRNA comes out of the nucleus it will contain: 1. No introns (intragenic regions that are not translated) 2. 3’ polyA tail 3. 5’ cap Then you can translate it. 3’ Poly A tail on mRNA The poly(A) tail is important for: 1. Nuclear export—if you have no poly A tail, you might not come out of the nucleus. Then you can’t get translated and can’t get the protein. 2. Proper translation—if you have no poly A tail, you might not be properly translated 3. Stability of mRNA—poly A tails prevent chewing of the end (prevent shortening) The tail is shortened over time, and, when it is short enough, the mRNA is enzymatically degraded FYI: This is the typical cap structure at the 5’ ends of eukaryotic mRNA Some proposed functions for the 5’ cap: 1. Regulation of nuclear export—need 5’ cap to help mRNA come out to nucleus 2. Prevention of degradation by exonucleases—5’ cap and poly A tail is doing similar things for mRNA 3. Helping translation 4. Helping in RNA splicing Pre-mRNA can’t be translated without removing inducer, 5’ and poly A tail. Regulation of gene expression by exonuclease activities A decapping enzyme can take off the cap and now it is exposed for degradation from that end. Protected at 3’ end by poly A and 5’ end by the cap. Remove those protections, and then you get chewed up. Want more protein: make more mRNA or make mRNA more stable. Increase transcriptionincrease mRNAincrease protein You can increase protein by decreasing degradation of mRNA (breakdown of mRNA) Can you make a cell express genes that it normally doesn’t? Hypothesis: Expression of neuron-specific transcription factors in fibroblasts will turn the latter into neurons Fibroblasts are found in connective tissue. They are involved in wound healing. Fibroblasts don’t have transcription factors to tell existing genes to make neuronal genes. The red pieces are three neuronal transcription factor genes They can be expressed in fibroblasts to make the transcription factors (proteins) that are normally not there. Now neuronal genes get expressed Conclusion: the expression of just three transcription factors is sufficient to transform a fibroblast into a neuron What does this do to the fibroblasts? Fibroblasts turn into neurons How could just three transcription factors do this? One transcription factor could affect many genes This means you can turn any cell into a heart cell Nice trick! Who cares? Cut lizard tail. Sox9 is a transcription factor that is involved in the regulation of cartilage development. No Sox9 was seen in the original tail, but Sox9 immunopositive cells were detected in the regenerating lizard tails (brown dots are Sox9 Some of the Key points 1. Regulation of transcription can control the amount of a gene product (protein) in a cell 2. This can be done by: A. Controlling the extent of DNA wound around nucleosomes B. Transcription factors and regulators—inducer vs repressors C. RNA processing and export—might not be properly exported thus improperly translated D. RNA degredation—make it hard to be broken down Could condensin affect gene regulation? When DNA is compacted, it cant be easily transcipted or expressed. Get rid of condensin--now gene is easier to be expressed. Translation From gene to protein Remember, there are also two other forms of RNA that can be made by transcription: rRNA tRNA Only mRNA is translated but translation machinery requires rRNA and tRNA. Only mRNA can be served as a template. Translation Looks like mRNA because theres poly A tail, 5’ cap and U The goal is to get amino acids in the right sequence and then release factor is put on to release it. The Genetic Code (these are codons) AUG codes for methionine(start codon) Do all DNA mutations change the amino acid sequences of proteins? NO!
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