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Biology 1002 Part 2 - midterm2.pdf

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Western University
Biology 1202B
Tom Haffie

Evolution of Eukaryotes February-18-13 2:31 PM Lecture 1. meaning of endosymbiosis, cyanobacteria, lateral gene transfer a. Endosymbiosis: a mutually beneficial relationship in which one organism lives inside another b. Cyanobacteria: microorganisms related to bacteria but capable of photosynthesis c. Lateral Gene Transfer: transfer of genes between organisms by a manner other than reproduction 2. origin of endomembrane system, nuclear membrane, ER etc. - Endomembrane system: the nuclear envelope and the endoplasmic reticulum: derived from infolding plasma membrane - Nuclear envelope separate genomic information from the rest of the cell, an advantage to tightly regulate transcription and replication of DNA separate from the rest of the cell  Also separates transcription and translation in space giving a level of sophistication not seen in bacteria 3. origin of mitochondria and chloroplasts - Surface area is important if this is where electron transport takes place - With a lot of energy produced by multiple mitochondria there are many different processes that can take place - PCG is expensive (75%) the endergonic process, this constrains the size of a prokaryotic genome since it does not have the energy to synthesize proteins - Constrainment of genome size is due to energy available - Mitochondria and chloroplasts are derived from anaerobic ancestral bacteria through phagocytosis 4. evidence supporting theory of endosymbiosis - Morphological: mitochondria are similar in appearance as bacteria, chloroplast look like that in cyanobacteria - Division: mitochondria/chloroplast divide similar to bacteria, genes do not produce mitochondria - ETC: only cells with ETC - Genomes: they have their own genomes - Transcription/translation machinery: have their own ribosomes 5. factors driving development of early eukaryotic cells a. Oxygen - The earliest bacteria were anaerobic: they used fermentation to keep glycolysis going, did not produce much ATP, not all of the free energy in glucose was obtained - Cyanobacteria can use oxygenic photosynthesis, they can split water, this creates O2 in the atmosphere - Leading to bacteria that can undergo aerobic respiration - ATP from oxygenic phosphorylation is much greater compared to fermentation making it more favourable b. Mitochondria/energy - The need for excessive amounts of energy demanded by increasing cell size (causing lower PM SA/V ratio) is met by mitochondria - The cell has enough energy to become more complex, can increase genome size 6. why eukaryotic cells can be larger and more complex than prokaryotic cells - In many bacteria protons are pumped out of the cell, they flow back in - There is a problem when oxygenic phosphorylation only takes place on the plasma membrane - A larger cell has more places for this to occur because there are more proteins and processes going on, there must be more energy to support this - Surface area increases, volume increases, as the cell gets bigger the volume increases faster than the surface area, this is a problem if the membrane is where electron transport and oxygenic phosphorylation occurs 7. evidence for lateral gene transfer from organelles to the nucleus - Mitochondria is a free living cell so there must be a symbiosis - Problem: coordinate the processes on the cellular level (glycolysis with what is happening in the - Problem: coordinate the processes on the cellular level (glycolysis with what is happening in the mitochondria) - Genes have moved from mitochondria to the nucleus - This puts the genes under tighter nuclear control - Genes still do the same thing but they are in a different organelle 8. general idea about how lateral gene transfer is detected (Southern bmtDNA nDNA - DNA on the membrane in a southern blot A B C A B C - Probe hybridizes to the gene - Isolate mitochondrial and nuclear DNA for 3 different plants - In species A lateral gene transfer has not occurred - In species B lateral gene transfer has occurred - In species C there is hybridization of both, both genomes have a copy of the gene 9. Hypotheses for why genes move to the nucleus from organelles (lateral gene transfer) - To coordinate processes on the cellular level (coordinating glycolysis with the mitochondria) 10. Possible reasons why certain genes have NOT moved to the nucleus from organelles - Eukaryotes that do not have mitochondria - Not evolutionary intermediates, they live in an environment where they do not need mitochondria - Clear evidence that they had them and got rid of them 11. Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes. - Cpn60 is a mitochondrial protein necessary for mitochondria to work, found in the nuclear genome, suggests that lateral gene transfer has occurred - Giardia has a copy of this gene - Lost mitochondria after lateral gene transfer Lecture 10 Summary March-12-13 3:45 PM 1. Evolution of Eukaryotes a. Oxygen - Once oxygen was available in the atmospherecell were able to utilize oxidative phosphorylationmaking much more energy available than from fermentation - Cells were able to becomemore sophisticated b. Mitochondria/Chloroplasts - Increasing cell size increased the demand for energy that could not be met due to electron transport taking place on the cell membrane - Phagocytosisof mitochondriaand chloroplast allowed for the cell to fulfill energy requirements and allow for a large more complex genome 2. Lateral Gene Transfer - Genes moving from organelles to the nucleus allowing coordinated cellular control - This is shown through: - Southern blotting showing that some cells have a mitochondrial gene in the mtDNA and some in the nDNA - A mitochondrialprotein found in Giardia, an organism that does not have any mitochondria,shows that it was once there but not any more Introduction to Prokaryote Gene Structure February-18-13 2:30 PM Lecture 1. relative sizes of typical mitochondrial, chloroplast and nuclear genomes - Nucleus: 120,000 kb (eukaryotes:linear chromosomes) - Mitochondrion: 16 kb - Chloroplasts:200 kb - Chromosomes in mitochondria and chloroplasts are circular and have many copies 2. rubisco structure and assembly from components coded by different genomes - Rubisco is a very abundantprotein important for carbon fixing in the Calvin's Cycle - Many protein complexesfound in the Calvin Cycle and the ETC are coded by genes that are found in both the chloroplast and the nucleus - When the protein undergoes transcription it uses mRNA, tRNA and ribosomes - Genes do not just code for proteins(PCG's) - They also code for mRNA, tRNA etc. 3. possible reasonswhy modern organelle genomes have become dramatically smaller over evolutionary time - Hosts in which their organelle genomehas suffered a mutation, where redundant genes are lost are selectively favored - Genes are also lost through lateral gene transfer (to the nucleus) - Some genes would be safely lost from the mitochondria because there is anothercopy in the nucleus Ex. Glycolysis genes would be deleted from the mitochondrial genome 4. possible reasonswhy genes have moved to the nucleus from organellesover evolutionarytime - Coordinative control - Organelles are involved in pathways generating ROS (oxygen + electron), they are reactive and mutagenic, putting DNA in nucleuskeeps it away from ROS - There is an entire gene expression inside mitochondriaand chloroplastsbut they cannotundergo sexual recombination and generate diversity 5. possible reasonswhy certain genes have not moved to the nucleus from organelles - The generalenvironmentin organelles is essentially prokaryotic, the environment in the nucleusis essentially eukaryotic, what has to happen to genes changing environments? - Local control? - Too much effort to transport to organelle? - Chance, not all the genes have moved yet? 6. basic mechanism of transcription and translation in prokaryotic organellesvs. eukaryotic nuclearenvironments - In prokaryotes RNA polymerase is the molecule that understandsthe information in a promoter,it bind to the cell and begins transcription - Complementarybase pairing about50 nucleotides a second in prokaryotes, reading 3' to 5' and synthesizing 5' to 3' - While this is occurring translation is also taking place - In Eukaryotes transcription must take place in the nucleus and the mRNA must enter the cytosol before translationcan take place 7. basic structure and function of RNA polymerase and ribosome RNA polymerase: reads 3' to 5' on the template strand,synthesizing 5' to 3' - A promoter sequence is understood by - The stop codon stops translation Ribosome: primarilyRNA machines - RNA is catalytic and the protein is structural - The RNA pairs with itself to create the 3D structure that they need to be catalytic 8. examples of complementary base pairing in gene expression 8. examples of complementary base pairing in gene expression - DNA with itself - mRNA with DNA - mRNA with itself - tRNA with itself - tRNA with mRNA - Ribosome, primarilyRNA with itself - rRNA in the SD box with mRNA E. Coli has about5000 genes in about 5000 kb of circular DNA One gene = 1 kb (1000 b) One amino acid = 3 bases About 300 amino acids in 5000 kb Different kinds of information in a DNA: - Codonsfor amino acids in proteins - RNA: mRNA, tRNA, - Promoter: where to start transcription,understoodby polymerase in prokaryotes - Terminator: where to stop transcription - Stop Codon: stops translation Lecture 11 Summary March-12-13 5:44 PM 1. Organelle Genomebecoming smaller a. Natural Selection:a genome losing redundant genes becomessmaller and selectively favoured b. Lateral Gene Transfer: genes movefrom the organelle to the nucleus - Coordinated control - The organelles are involved in producing ROS, the nucleus keeps the DNA away from ROS - No sexual recombinationin organelles c. Lost safely because there is a copy in the nucleus (glycolysisgenes) 2. Information in DNA - Codons for amino acids in proteins - RNA: mRNA, tRNA - Promoter:where to start transcription, understood by polymerasein prokaryotes - Terminator:where to stop transcription - Stop Codon: stops translation 3. Transcription and translation in different environments - In eukaryotestranscription and translation are spatially separated - In prokaryotesthere is no spatial separation and translation occurs as transcription is occuring Prokaryotic Gene Function February-18-13 2:36 PM Pre Lecture 1. identify the sequence of standard "start" and "stop" codons - The template strand for a given gene is always read 3' to 5' Start: AUG STOP: UAA, UAG, UGA 2. identify the function of "start" and "stop" codons - Start codon: the first codon read in mRNA translation - Stop codon (nonsense or terminating codon): does not specify for an amino acid, when a ribosome reaches one of the stop codonspolypeptidesynthesis stops and the new polypeptide chain is released from the ribosome 3. compare the overall gene expression of prokaryotic vs. eukaryotic cells. - The process of transcription and translation are similar in both cells - Prokaryotes can transcribe and translate a give gene simultaneously - Eukaryotes transcribe and process mRNA in the nucleus before exporting it to the cytoplasm for transcriptionon ribosomes Lecture 1. relative location of such DNA sequence “signals” as promoter,5’ and 3’ UTR, “SD box”, start codon,stop codon, transcriptionterminator etc. UTR (Untranslatedregion)- region of DNA, transcribed into mRNA that is not translated because it is upstream of the start codon - tRNA does not pair with the UTR however, rRNA will pair "SD" Box- a region on the DNA that once transcribed into mRNA, base paired with rRNA to help with the initiation of translation 2. mechanism by which each signal is interpreted,or understood,by the cell a. Transcription: Promoter Sequence- controlling element for transcriptional elements of genes, attract RNA polymerase - Understood as DNA Terminator Sequence- is transcribed into mRNA and makes a stem loop structure, pairing with itself, destabilizing mRNA causing it to dissociate from DNA - Understood as RNA b. Translation: Start Codon- tRNA binds to correspondingnucleotide sequences - Understood as RNA Stop Codons- tRNA does not bind to stop codons, a termination release factor gets into the active site in the translating ribosome due to no competing tRNA - The release factor is a protein so does not base pair with the mRNA 3. relationship between DNA sequence of signals and their function (ie. how would low efficiency promoters be different than high efficiency promoters? - Promoters have a general common structure or sequence but are variable, they vary in attractiveness and their ability to drive transcription  Understood as DNA - Terminators create an mRNA loop, G-C bonds are more stable than A-T bonds, for a more stable mRNA loop it would require more G-C bonds  Understood as RNA 4. characteristics of promoters that require a particular position and direction - The promoter sequence is where polymerase will bind to the DNA for transcription - Where the polymerase binds and the direction it binds will determine what genes are transcribed since polymerase must read the DNA 3' to 5' - There is something important at -10, something important at -35, this gives promoters direction - For a given chromosomethere is not strand that is template all the time, each gene might be coded on one strand or the other, depending where the promoter is 5. change in amino acid coded, given a change in the DNA sequence(and Genetic Code table) - Since the code is universal the coding run on for transcription in the nucleusand translation in the cytoplasm must be the same - This is proven by lateral gene transfer from organelles such as the mitochondriato the nucleus - Some amino acids have multiple codons that code for them 6. base sequenceof start and stop codons as mRNA and DNA Start Sequence: mRNA: AUG DNA: TAC Stop Sequence: mRNA: UAA, UAG, UGA DNA: ATT, ATC, ACT Clicker Question: 1. Which DNA strand will be the template for Gene b? There is not enough information to know which strand will be the template, you need to know where the polymerase is (the promoter), if the promoter is on the right it will read 3’ to 5’ on the top, this is the strand that will be read If polymerase binds on the right it will be the top strand, if the left it will be the bottom strand - For a given chromosomethere is no strand that is template all of the time, each gene may be coded on one strand or the other, just depends on where the promoter is 2. The “start codon” is the…. a. Signal for start of transcription Wrong: translation b. Same in prokaryotes and eukaryotes c. First three bases of mRNA Wrong: many bases are transcribed before the start codon d. Sequence 3’ “TAC” 5’ in template Prokaryotic Gene Regulation February-24-13 3:14 PM Pre-Lecture 1. identify the main features of bacterial operons Operon:a cluster of prokaryotic genes and the DNA sequencesinvolved in their regulation - Promoter: where RNA polymerase binds for transcription - Operator: a short segmentthat is a binding sequence for a regulatory protein - Transcription Unit: a group of genes in the operon that are transcribed into mRNA  Operons can contain many genes and are transcribed as a unit, therefor the single mRNA can code for many proteins 2. identify the function of repressor proteins - Repressor:a regulatory protein that controls an operon, when bound to the DNA, reduces the likelihood that genes will be transcribed - Activator:a regulatory protein that controls an operon, when bound to the DNA, increasesthe likelihood hat genes will be transcribed  Operons can be controlled by more than one regulatory mechanism  A repressor or activatorcan control more than one operon 3. identify location of various components of the lac operon Regulatory gene Transcription unit of three structural genes lacI lacZ lacY lacA Promoter Operator DNA Transcription initiationion termination site site Lac repressor β-Galactosidase Permease Transacetylase RNA polymerase Lecture 1. DNA signals in RNA-codinggenes - What signals are transcribed into mRNA from DNA Promoter SD Box Start Codons Stop Terminator a. Start Codon- transcribe and also translated - In mRNA and protein b. Stop Codon- transcribed but there is no tRNA for a stop codon, it is not translated - In mRNA c. Promoter Sequence-attract the attention of polymerase but are not transcribed, the +1 nucleotide is downstream - Not in mRNA d. Terminator Sequence-transcribed but is not translated, it is past the stop codon - In mRNA e. SD Box- is transcribed,base pairing with rRNA to help initiate translation - In mRNA 2. DNA sequence of anticodon in tRNA gene, given the codon - In a tRNA gene there is only a promoter and a terminator sequence,there are no codons - The DNA sequence gets transcribed into tRNA and base pairs complementarywith mRNA 3. likely effectof base sequence substitutions in various DNA signals a. Promoter- effect on transcription depends, it could cause an increase or decreasein affinity to polymerase b. SD Box- effecton translation depends, it could cause an increaseor decrease in affinity to rRNA c. Start Codon- translation cannot proceed, there is no way to change a start codon without breaking it d. Stop Codon- effecton translation depends, there are 3 differentstop codons - If altered to another codon, translation will not stop until it finds another stop codon e. Terminator- effect on transcription depends, the ability for mRNA to pair with itself and createa stem-loop structure 4. changein amino acid coded, given a changein the DNA sequence(and Genetic Code table) a. Silent Mutation: one mutated base pair = No changein amino acid coded for b. Missense Mutation: one mutated base pair = Change in amino acid coded for c. Nonsense Mutation: one mutated base pair = Change in amino acid coded for to a stop codon d. Indel Mutation: insertion of one base pair = Change in multiple amino acids coded for, shifting the reading frame 5. base sequence of start and stop codons as mRNA and DNA - Codons on DNA are coded 3' to 5' but are understood as mRNA 5' to 3 a. Start Codon - DNA: TAC coded 3' to 5' - mRNA:AUG understood 5' to 3' b. Stop Codon - DNA: ATT, ATC,ACTcoded 3' to 5' - mRNA:UAA, UAG, UGA understood 5' to 3' 6. the location of various signals given a diagram of gene expression Promoter SD Box Start Codons Stop Terminator 7. mechanismof action of lac repressor Promoter lacI Operator lacZ lacY lacA DNA Transcription blocked 1 mRNA 3 2 Lac repressor (active) - When the lac repressor binds to the operator DNA (positively chargeprotein + negatively charged DNA) polymerase cannot transcribe it - 2 molecules often form a dimer and bind together, in the lac operon two moleculesof lac repressor bind to the operator on either side of a portion of DNA, this loop prevents polymerase from transcribing the DNA 9. function of lac operon in the presence,and absence, of lactose lac operon lacI Promoter Operator lacZ lacY lacA - 2 molecules often form a dimer and bind together, in the lac operon two moleculesof lac repressor bind to the operator on either side of a portion of DNA, this loop prevents polymerase from transcribing the DNA 9. function of lac operon in the presence,and absence, of lactose lac operon lacI Promoter Operator lacZ lacY lacA DNA Transcription occurs RNA 5 mRNA polymerase binds to 4 mRNA Lac promoter Inactive Translation 6 repressor repressor (active) 3 Binding Allolactose Lactose site for (inducer) catabolism inducer enzymes 2 1 Lactose - In the presence of the lactose the transcribed lac repressor binds with allolactose which inhibits binding to the operator - RNA polymerase binds to the promoter, transcription occurs,translation occurs 10. possible location of mutations in lac operon that give rise to a given phenotype - Mutations in the promoter vary the attractivenessto RNA polymerase - Poly-systronic message - Lac I: produces Lac repressor 11. phenotype that would arise from a given mutation in lac operon under given conditions NOTE: lac operon regulation is also sensitive to glucose levels through binding of cAMP/CAPas summarized in Fig. 14.4. This aspect of lac regulation is NOT part of the course. Eukaryotic Genes March-04-13 8:24 PM Lecture 1. basic structure of eukaryotic vs prokaryotic cell with respect to gene expression - With respect to gene expression DNA is transcribed within the nucleus in Eukaryotes, transported outside the nucleus and then translated - In Prokaryotes this process all takes place in the cytoplasm where translation can begin while transcription is still occurring 2. structure of eukaryotic endomembrane system with respect to gene expression - Endomembrane proteins have an amino acid tag that tell the cell they are required for co-translational support in the endoplasmic reticulum - Amino acid tags are most likely located on the coding region of the gene 3. structure of eukaryotic promoters/enhancers Promoter proximal Enhancer region Promoter Transcription unit of gene Exon Intron Exon Intron Exon DNA Regulatory Promoter TATA 5 ' UTR 3 ' UTR sequences proximal box elements (regulatory sequences) - In Eukaryotes it is polymerase II that recognizes promoters, within the promoter there is a region called the TATA box - The TATA binding protein associates with the promoter making it more attractive, naked proteins are not as attractive to polymerase II - There are also other proteins that are attracted to the promoter Coactivator (multiprotein complex) - Activators on both the enhancer and the promoter proximal Transcription region bound together through a coactivator (multiprotein initiation site complex) Activator Activator - Having the enhancer interact with the promoter makes the Enhancer Promoter promoter more attractive proximal Promoter - Enhancers are direction and location independent, DNA will fold region and loop in order to bind to the proximal region DNA loop Maximal transcription 4. protein motifs common in DNA binding proteins - Electrostaticly, the DNA is negatively charges along the backbone and proteins can be Helix Turn positively charged major groove - Only works if the protein has a particular shapes that fits into the double helix - The lac repressor has a helix-turn-helix shape Zinc ion COO– - Some proteins that bind DNA are able to do that because they have zinc fingers in Finger 3 Finger 2 the proteins Helix in major groove Zinc ioCOO– - Some proteins that bind DNA are able to do that because they have zinc fingers in Finger 2 Finger 3 the proteins - Ex. If you find a gene sequence you would be able to point a zinc finger, a sequence that would fold around zinc, predicting that it is a DNA binding protein, becauase it Finger 1 codes for a zinc binding site N3+ Leucine zipper - Coding for a DNA binding protein if it contains a leucine zipper region 5. mechanism of transcription termination in euks - Eukaryotes do not have loops in the mRNA, polymerase transcribes through the polyadenylation signal, the signal is recognized in mRNA by RNAse, that cleaves it - Polymerase stops transcribing when the mRNA is cut - When it is cut you add the poly (a) tail, there is no base pairing, added by… 6. mechanism of translation initiation in euks - The Met-tRNA+GTP+ribosomal subunit complex binds to the 5’ cap of the mRNA and moves along the mRNA scanning until it reaches the AUG start codon in the P site - Base pairing occurs between the codon and the anticodon of the initiator Met-tRNA 7. which gene expression components cross the nuclear membrane to get from where they are made to where they function - Nuclear proteins have an amino acid tag that tells the cell to send the protein to the nucleus through pores in the nuclear membrane 8. various stages of gene expression subject to regulation a. Transcriptional regulation: Determines which genes are transcribed - Regulation of transcription initiation - Chromatin remodeling to make genes accessible for transcription b. Posttranscriptional regulation: Determines types and availability of mRNAs to ribosomes - Variations in pre-mRNA processing - Removal of masking proteins - Variations in rate of mRNA breakdown - RNA interference c. Translational regulation: Determines rate at which proteins are made - Variations in rate of initiation of protein synthesis d. Posttranslational regulation: Determines availability of finished proteins - Variations in rate of protein processing - Removal of masking segments - Variations in rate of protein breakdown 9. how organisms express different genes in each different tissue - Regulating enhancers (attractiveness of promoters) - Tissue specific activator proteins, some proteins would only be produced in your eye, therefor only able to enhance genes expressed in your eye, the same goes for different tissues 10. the advantages to alternative splicing - In some tissue a certain gene may be an intron and in another tissue it may be an exon Introns are removed through: ○ Introns attract snRNPs, a complex of protein and RNA, small nuclear RNAase, they are paired with themselves with themselves ○ snRNPs make a spliceosome ○ snRNA complementary base pairs with the message, there are signals in the DNA, they are transcribed into RNA and understood by snRNPs ○ The intron is looped out and removed - One gene can give rise to many different proteins through this choice of alternative splicing - Neurons want to make connections with other neurons and not itself, each neuron needs an identity - One gene makes 40,000 different proteins from the same message through alternative splicing - Because they can keep their RNA away from the ribosomes eukaryotes can accomplish this 11. mechanism of action of miRNA - There are genes that code for RNA called micro RNA (miRNA), they are transcribed and pair with themselves, they come out of the nucleus and encounter a protein called dicer resulting a linear molecule that binds to the 3’ UTR of another gene - Prevents translation, a surgical way to stop the expression of a particular gene 12. mechanism of targeting proteins to cellu
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