Biology 1002 Midterm 2 Notes covering Lectures 10-17.pdf

Bio Lecture 10-17 Lecture 10- Evolution of Eukaryotes • from a morphological point of view eukaryotes are very complex • chlammy is a classic eukaryotic system • bacteria and archaea are not morphologically complex • their cells are small biochemically they do some interesting reactions (one gene, one enzyme) • • morphology: cellular complexity What was it that changed at the biforkation of the tree of life? The Paradox • all morphologically complex life is eukaryotic • all eukaryotes share common complex traits • Nucleus, trafficking, cytoskeleton, sex phagocytosis (the ability to bring stuff in from the outside) , organelles and the more complex ones have tissue types, different cells (plants have different cells too) • these are all morphological features of a eukaryote • Prokaryotes show no tendency to form this type of morphological (cell) complexity Why don’t they if evolution is stepwise? • • evolution is gradual( ex. the eye) but it seems quite that eukaryotes had this new complex ability What drove the evolution of eukaryotes? • OXYGEN IS THE KEY • the driving element of eukaryotic life is the ability to use O2 • earliest bacteria were anaerobic • 4 billion years ago=anaerobic • anaerobic= uses fermentation fuels the ability to keep glycolysis going but this doesn’t give us much ATP • breakdown Glucose but you dont harness the free energy • these guys are starved for ATp • Anaerobic • (1) Not requiring, or capable of occurring, in the absence of air or free oxygen. • (2) Caused by, or relating to, the lack of molecular oxygen. • Anaerobic may be used to describe an organism, a cell, a process or a mechanism that can function without air (i.e. air to generally mean oxygen). This is in contrast to the term aerobic, which means requiring air or free oxygen. Bio Lecture 10-17 • ABOUT 2 BILLION YEARS AGO- development of cyanobacteria •they are prokaryotic but can do oxygenic photosynthesis (can split water to O2) •this ability lead to O2 in the air •they have PS2 and PS1 •only prokaryote that have a photosynthetic complex like the chloroplast they have oxygenic photosynthesis, they have an O2 evolving complex next to PS2 • THIS LEAD TO O2 IN THE AIR AND BACTERIA THAT UNDERGO AEROBIC RESPIRATION • earliest bacteria were anaerobic--->2.2bya get cyanobacteria (Oxygenic photosynthesis)---> bacteria that undergo aerobic respiration • aerobic respiration= oxidative phosphorylation •could only develop after O2 was in the air •highly electronegative (+ redox potential) •terminal electron acceptor of respiration (ETC) •huge amount of ATP from oxidative phosphorylation compared to gylcolysis and fermentation Oxidative Phosphorylation in Bacteria • bacteria and archaea are thousands of times smaller than eukaryotes have a cell and ox phos ETC, chemeosmosis of ATP, synthesis of the breakdown of • glucose its occurs on the plasma membrane • thats the membrane on the bacteria • this is the membrane used for OX PHOS • actually in bacteria protons used in ATP synthase are pumped out of the cell and then harnessed for ATP synthesis by being pumped back into the cell •pumping occurs outside the cell and then they are pumped back in • there’s a problem with having OX PHOS in the membrane • when a bacteria is bigger you have more centers of OX PHOS more ETC’s on the plasma membrane, you need more of them because the volume of the cell goes up • small cell means fewer number of proteins, fewer number of energy demanding/ energy requiring reactions in the cell • when you get bigger you need more energy to support yourself • Area= 4Pi 2 • volume= 4/3 Pi 3 so as the cell gets bigger the volume increases much faster than the surface area of • the membrane does • this is a problem if your OX PHOS and ETC are in the membrane of the cell • this is what limits the size of a bacteria; they can’t support an ever increasing cell volume • there comes a point where you cannot fill the membrane with enough OX PHOS sites to reach the demands of the cell Bio Lecture 10-17 Eukaryotes have much bigger genomes • eukaryotes (10 to the 9 or 10 to the 10) are a million fold bigger bacteria • ten thousand times larger than bacteria • huge diversity of genome size in a eukaryote when prokaryotes are relatively narrow this is constrained by the amount of energy a prokaryote can have to devote to protein • synthesis • some plants have much bigger genomes than humans • genome size varies greatly amount eukaryotes because they have lots of ATP available •not constrained to have a small genome Eukaryotic Cells-more energy! • high PM surface area/volume • eukaryotes have a low PM surface area/ volume ratio but it doesnt matter because their OX PHOS doesn’t occur in their plasma cell • they have mitochondria •liver cell in human have 2 thousand mitochondria, each having many OX PHOS centers membrane area internally in the cell is gigantic versus the plasma membrane • • now lots of energy due to lots of inner folding in mitochondrial matrix which means lots of sites of ETC and OX PHOS • NOW LOTS OF ENERY= SUPPORT LARGER CELL (LARGER GENOME) • replication of DNA doesn’t take much energy • but proteins synthesis does • DNA--> protein (energonic process) takes up 75% of cell energy • this also confines prokaryote •doesn’t have enough energy to make many proteins so it only has a few • eukaryotes have many more proteins (this underlies the complexity of eukaryotes) • a typical bacterium has 13000 ribosomes the center for protein synthesis • a typical eukaryote has 10000 times more ribosomes Bio Lecture 10-17 Endosymbiosis • • happened about 2 billion years ago • we don’t know much about the progenitor of the eukayrotic cell was • was it bacteria? archaea? • most likely ancestral bacterium • an ENDOMEMBRANE SYSTEM must evolve • one of the defining characteristics of eukaryotes is the nuclear envelope, the infolding of PM • Endomembrane system: ER attached to the nuclear envelope are derived from infolding of the plasma membrane • every eukaryote has a nucleus • which is advantages because it separates genomic information from the rest of the cell having a nuclear envelope to control transcription and translation of DNA seperate • from the rest of the cell allows you to tightly control transcription in ways bacterium can’t • can have specialized transcription factors, tightly regulated transcription and translation which are separated by space which gives you a level of sophisticated control that you don’t see in bacterium • Endosymbiosis: that mitochondria and chloroplast are derived from free living prokaryotic cells • The evolutionary process of endosymbiosis created organisms with typically "prokaryotic" genes in organelles and typically "eukaryotic" genes in the nucleus • mitochondria from bacteria • this bacterium was probably anaerobic, advantage (through phagositosis) to bring in this type of bacterium to supply you with lots of ATP • Cyanobacterium have PS1 and PS2 have identical processes to the chloroplast which gives more evidence chloroplast is descendent from an ancient cyanobacterium (2 billion years ago) • • all eukaryotes have mitochondria which means this endosymbiosis of this anaerobic bacterium predated the uptake of the cyanobacteria • top branches get you plants and algae • and the lower branch gives you animals and fungi ( see diagram above) • anaerobic was picked up first then in a subset of those, cyanobacteria • Bio Lecture 10-17 Evidence for Endosymbiosis 1. morphology (mitochondria look like bacteria) (chloroplast look like cyanobacteria) 2. formation/divison (when mito’s and chloroplasts divide they look like bacterium dividing) 2.1. cant make a mitochondria( no gene that codes for it) 2.2. can only get mito from old mito dividing 3. electron transport chains ▯ 3.1 mito and chloroplasts are the only organelles within eukaryotes that have electron transport chains 4. Genomes ▯ 4.1. they have their own genomes ▯ 4.2. if they were once free living cells they would need their own genomic information and they do 5. Transcription/translation machinery (same as 4.2 have the machinery for DNA coversion) ▯ 5.1 have ribsomes Lateral Gene Transfer • green is nucleus • yellow is mito (see lecture slide for better picture) • Problem? Who’s the boss? • Mito that are free living and have a big cell that compartmentalizes it genomic info (nucleus) • mito and nucleus both want to do things • need a symbiosis (mito cannot just be free and do what it wants) • mito must become a part of the eukaryotic cell • Problem: to coordinate the processes on a cellular level • coordinate glycolysis with whats going on in the rest of the cell • over millions of years, the organelle genes have moved to the nucleus this is called lateral gene transfer of horizontal gene transfer • • lateral gene transfer: genes from mito and chloro relocate to nucleus which puts them under a tighter nuclear control Bio Lecture 10-17 • nucleus gained the upperhand and now controls the organelles • complex 1 has 40 proteins, some genes are not coded by mito anymore but those genes have been shipped off to the nucleus, proteins still do what they always did! • no deletion of function just the location of the genetic info is different Southern Blot- Detecting Lateral Gene Transfer • very useful for determining if a genome has a particular gene or the number of copies of that gene • guy named Southern developed this • just genomic DNA • run it on a gel and make it so DNA is single stranded on membrane • make DNA single stranded probe • then the probe will hyridize • hybridization occurs that means that on the membrane their is a DNA sequence similar to that of the probe • you can identify the amount of a gene or the number of copies • can isolate DNA from the mitochondrial genome and nucleus DNA • lateral gene transfer is still occurring today • compare similar species • has lateral gene transfer occurred in one species and not the other? maps lateral gene transfer • • ex. species A transfer hasn’t occurred yet • lane be, transfer as occurred • lane c transfer is still occurring • position on lane doesn’t matter, JUST PRESENCE OR ABSENCE • see lecture slide for diagram • make a copy, send a copy to the nucleus, then the original is destroyed in either the mito or the chloro Mitochondrion-less Eukaryotes • there are eukaryotes that don’t have mitochondria they cause diseases in humans • EX. Giardi trichomonas, get in your gut and cause sickness • thought they were evolutionary intermediates between prokaryotic and eukaryotic cells WRONG • • they just lack mitochondria because they are in an environment which allows them not to have mito • they use fermentation and have a very protective environment • they did have mito but they got rid of it Bio Lecture 10-17 What is Giardia doing with cpn60? • cpn 60 is a mitochondrial protein • without it you die • essential for mito to work • cpn60 is in the human genome mitochondrial protein that suggests that lateral gene transfer has occurred • • cpn60 is just not found in the mito it is found in the nucleus • Giardia has a copy of cpn60 • the ancestor of all eukaryotes shows that lateral gene transfer happened very early on because both Giardia and other eukaryotes have a copy of cpn60 in the nucleus this gene cpn60 has no function in Giardia Lecture 11: Intro to Prokayotic Gene Structure (when reviewing reread chapter 13 and 14) Evolution of eukaryotes: Endomembranes and Endosymbiosis • important Mordern Chlammy cells have 3 different genomes 1.nuclear 2.mitochondrian 3.chloroplast Modern Endoymbiont genomes are greatly diminished • 120000 kb in the nuclear membrane of Chlammy and 17 linearly chromosomes • chloroplast has 200kb • mitochondrian 16kb • the chromosomes in the chloroplast and mitochondria are circular and present in many copies of the organelle chromosomes E.coli has about 5000 genes in about 5000 kilobases of circular DNA assume that the E.coli genome is similar to the ancient prokaryotes that gave rise to • chloroplasts and mitochondria • so about 5000 genes • typical protein in E.Coli? about 300 • one gene is about a thousand base pairs (1 kb) • and each amino acid needs 3 bases to code, each codon is 3 bases • about 300 proteins in a prokaryote of this size • know how to do this math Bio Lecture 10-17 Vaucheria (algae) haas only 169 genes in only 115 kb of circular cpDNA • this genome has gone from 5000 to 115kb over evolutionary time Humans have only 37 genes in only 16 kb of circular mtDNA (mitochondrial genome) • but only make about a dozen proteins • but 37 genes • why more genes than proteins??? • what do genes code for if they dont code for proteins? • all RNAs are made by transcription of genes • their is a gene for each tRNA and rRNA • gene could be a protein coding gene or an RNA coding gene • 37 genes in mito but lots are coding for RNA Modern Endoymbiont genomes are greatly diminished WHY THE DIMINISH FROM 5000 KB TO 37???? WHY WOULD A CELL STICK ITS MITO GENES IN THE NUCLEUS? • having two copies both in the nucleus and the mito/chloro is redundant • some of these genes that were lost are: • genes that are used for free living organisms but are not needed in organelles • genes that code for flagella • hexicinase • genes for gylcolysis • don’t need these genes in the mitochondria when its already in the nucleus • genomes are smaller because some genes have been deleted • language of evolution, hosts in which the mito has suffered a mutation/deletion such that it has lost a bunch of genes makes it easier to replicate such genes • these organisms would have a selective advantage • one way to get rid of them is mutation and deletion • another way is lateral gene transfer • why would genes transfer to the nucleus? so the nucleus has more coordinated control • • overts cellular rejection • these organelles have ETC and oxygen metabolism that generate ROS • O2 plus an electron gives us ROS • these organelles are sites for ROS production • ROS is very reactive and very mutagenetic • so the genes are moved away from the ROS into the nucleus • what can genes do in the nucleus that they can’t do in organelles? sexual recombination! • • note: organelle genomes also have machinary for transcription and translation Bio Lecture 10-17 • gene expression machinery and even ribosomes are in the organelle • Why haven’t all organelle genes moved to the nucleus? • maybe they need local control (be close to sense a problem) • maybe its too much hassle or it takes too much time to relocate, too hard to transport • too big to move • maybe they don’t work in the nucleus many its disadvantageous to move it, its in its optimal position • • maybe its just chance (genetic drift) • maybe there hasn’t been enough time yet, maybe in the future we’ll have less • Bombtime: the general environment in organelles is prokaryotic • The evolutionary process of endosymbiosis created organisms with typically "prokaryotic" genes in organelles and typically "eukaryotic" genes in the nucleus. • so what has to happen to genes to move from a prokaryotic structure to a eukaryotic one? “Solar-powered” sea slugs steal chloroplasts from algae! • ELSYIA • as a juvenile it feeds on the algae vaucheria • coenocyt: doesn’t have cell walls • SO, elsyia sucks the guts out of the vaucheria algae guts contain chloroplasts • • elsyia takes those chloroplasts and incorporates those chloroplasts in cells along its digestive track • this animal brings chloroplasts inside its own cells and then elysia stops eating and lives off photosynthetically reduced carbon for the rest of its life • its an autotrophic animal, photosynthetic animal • its lives for ten months • its lives off photosynthesis for ten months • Blue proteins of ETs and Calvin Cycle are coded by genes that are no longer in the chloroplast DNA sequence as Music • in S2 translation, the DNA coding sequence for the S2 protein (a membrane receptor for the neurotransmitter serotonin) was convered to music that plays out both the DNA sequence and the sequence of the encoded protein. They assigned the notes C,A,G, and E to the bases cytosine,adenine, guanine, and thymine. Under this melodic line, the bass progressions are structured to reflect the characteristics of the encoded amino acids, inclduing their water-solubility, charge, and size. Higher-order structure of the protein is suggested by changes in tonality. • WTF ITS ACTUALLY MUSIC! Bio Lecture 10-17 • mRNA • diagram shows that mRNA pairs with itself • complementary base pairing!!!!!! • IT FUCKING PAIRS WITH ITSELF! NO FUCKING WAY • This is all complimentary base pairing • tRNA pairs with itself to gain its proper 3D structure • tRNA also pairs with mRNA in the anticodon Bio Lecture 10-17 • rRNA base pairs with itself to form 3D shape and is catalytic • rRNA are primarily RNA, machines • RNA is catalytic and the protein is structural • Protein: DNA polymerase (representative protein) • the DNA polymerase gene has figured out how to replicate itself that gene codes for a protein that makes more of the gene • Bio Lecture 10-17 • codons that specify for amino acids are information in DNA • promoter: where to start transcription • in prokaryotes RNA polymerase understand promoter • (different kinds of info) Bio Lecture 10-17 • adding complementary base pairs (transcripting) • prokaryotes: about 50 nucleotides per second are being laid down by polymerase reading 3’ to 5’ • • synthesizing 5’ to 3’ • stop codon signals the end • stop codons stop translation!!!! • dont stop transcription • RNA polymerase does not understand the genetic code Bio Lecture 10-17 Lecture 12: Prokaryotic Gene Function Modern endoymbiont Genomes are greatly dimished • the environment of a nucleus and an organelle is very different • genes and how they are expressed are different • organelles are bacterial nucleus is eukaryotic • • MRNApairs with itself • tRNAs pair with themselves • DNApolymerase gene copies itself Bacteria transcription initiates the Promoter sequence • Promoters attract DNApolymerase • a bubble forms and makes the DNAsingle stranded • ATAT rich sequence at about -10 and -35 • -ive starts from the actual startpoint • template strand is getting transcripted • “downstream” from startpoint(beginning of bubble). • black arrows are where the DNApolymerase touches the DNA • other arrows are indications of locations where mutations can affect the effectiveness of the promotor • almost all genetic regulations are like dimmer switches • some promoters are very attractive and polymerase binds very stably and initiates transcription frequently other promoters are less attractive and efficient • • so! promotors have similar structure but are also very different (different transcription rates) • How does transcription in bacteria terminate? How does polymerase know its at the end of the gene? • sequence of DNAcalled terminator sequence which gets transcripted from the DNAto the mRNA • it then makes a loop structure by pairing with itself! • that loop signals polymerase to stop! • THE LOOP CAUSES THE MRNATO DISASSOCIATE FROM THE DNA • terminator sequence is in DNAbut only understood in RNA • the signals are all in DNAbut signals are understood differently Bio Lecture 10-17 • example!... a particular terminator stops transcription 60% of the time and another one that stops it 40% of the time! • WHATS THE DIFFERENEC? • maybe the length of the terminator sequence is longer which makes more stable which makes the termination precent higher • maybe the forces of the attraction are higher (G and Cs have 3 H2 bonds so pack the terminator with G and Cs to make it more stable) Bacterial Transcription Terminates a Hairpin Loop • in order to know what is a template strand you need to know where the promotor is • promotors give direction, when DNApoly. binds to a promotor they must go the way they are told • which ever strand is 3’to 5’will be read by polymerase • for a given chromosomes there is no template strand each time, each one can be used depending on where the promotor is • the start codon is the same in prok and euk cells! • start codons are only understood in the mRNAbut it is in the DNA • in RNAthe start codons areAUG so in DNA3’to 5’, TAC • start codons dont start transcription and the it is not the first 3 bases of mRNA( many bases are transcribed b4 the start codon) • START CODONS STARTS TRANSLATION • 5’to 3’UTR, is the bases transcribed b4 the start codon Bio Lecture 10-17 •untranslated region! it is upstream of the start codon Ribosomes have 2 subunits, each containing rRNAand Proteins E = exit site P = peptidyl site A = aminoacyl site (from left to right) • translation initiation is stabilized by mRNA/rRNAbase pairing • start codon attracts Met. @ site P and that initiates process of translation • bases to the left of the start codon is the UTR • those bases are reaching up waiting to pair but the UTR is not paired with anything • except no tRNAwill pair with them • in bacteria there is rRNA • rRNAwill pair with the mRNAand that sequence is called the SD BOX • IT ISAREGION ON THE DNATHAT WAS TRANSLATED INTO MRNAAND THEN BASE PAIRED WITH RRNATO HELPTHE INITIATION OFTRANSLATION Aprotein Release Factor Terminates Translation • there is no tRNAthat binds to stop codons and the protein binds to theAside • the release factor is always trying to get in there but is outcompeted by tRNAexcept for the stop codon where there is no tRNA • protein does not base pair with nucleic acid! Impossible! genetic code is universal • •need to be if lateral gene transfer was going to work • some amino acids have many codons Bio Lecture 10-17 • proks don’t have free floating mRNAwhile euks do • proks cells mRNAs begin to be translated even b4 they are finished being made • as soon as mRNAis available and the SD box is transcribed, the ribosomes start translation • so transcription and translation can occur at the same time • polymerases are transcribing the same gene at the same time and as fast as they can • as fast as the promotor will allow this makes massive amount of protein product • • faint line is the template strand and the beads on the string are ribosomes! • which ribosome has been translating the longest? the longest polypeptide Bio Lecture 10-17 • which way are the RNApoly. travellng? • Which way are the ribosomes travelling? Lecture 13: Prokaryotic Gene Translation Assignment Reading Notes: Chapter 14 14.1a: The Operon Is AUnit of Transcription • form typical metabolic process,several genes are involved and they must be regulated in a coordinated fashion • the on/off control of these genes is at the level of transcription operon model applies to the regulation of genes in bacteria and their viruses • • Operon: is a cluster of prokaryotic genes and the DNAsequence involved in their regulation. • Promoter: is a region where the RNApolymerase begins translation • Operator: a short segment that is a binding sequence for a regulatory gene • Regulatory gene: a gene that is separate from the operon encodes the regulatory protein • Repressor (regulatory protein): when bound to the DNA, reduces likelihood that genes will be transcribed • many operons are controlled by a number of repressors or activators which allows for superimposed controls that provide regulation of transcription, allowing for instantaneous global responses to changing environmental conditions • Each operon, which can contain several to many genes, is transcribed as a unit from the promoter into a single messenger RNAand as a result the mRNAcontains codes for several proteins Transcription Unit: a cluster of genes transcribed into a single mRNA • ribosome translates the entire mRNAfrom one end to the other, making each protein encoded in the mRNA the proteins encoded by genes in the same operon catalyze steps in the same process, such as • enzymes acting in sequence in a biochemical pathway 14.1b: The lac operon for Lactose Metabolism is Transcribed When an Inducer Inactivates a Repressor • lacZ,lacY,lacA(genes) are adjacent to one another on the chromosome in the order-Z-Y-A • they are transcribed as a unit into a single mRNAstarting with lZ • lZ encodes for the enzyme B-glalactosidase, which catalyzes the conversion lactose into glucose and galactose Bio Lecture 10-17 • these sugars are further metabolized by other enzymes producing energy for the cell by glycolysis and the Krebs cycle • lY encodes a permease enzyme that transports lactose actively to the cell • lAgene encodes a transacetylase enzyme, the function of which is more relevant to metabolism of compounds other than lactose • Lac Operon: cluster of genes and adjacent sequences that control their expression • the operator for the Lac operon is a short DNAsequence between the promotor and the lacZ gene • lac operon is controlled by a regulatory protein that is called the lac repressor • lac repressor is encoded by the regulatory gene lacI, which is separate from the lac operon Lactose absent from medium: structural genes expressed at very low levels 1. Active lac repressor expressed from lacI binds to operator 2. RNApolymerase blocked from binding to operator 3. Transcription of structural genes occurs rarely. (repressor occasionally falls off operator, allowing a very low rate of transcription, resulting in a few molecules of each enzyme made) *Repressor binding is a kind of equilibrium; while it is bound to the operator most of the time, it occasionally comes off. In moments when the repressor is not bound polymerase can successfully transcribe.As a result, there is always a low concentration of lac operon gene products in the cell. Lactose present in medium: structural genes expressed at high levels 1. Permease molecules already present transport lactose into the cell 2. B-galactosidase molecules already present in the cell convert some of the lactose to the inducer allolactose 3. Allolactose binds to the lac repressor, inactivating it by altering its shape so that it cannot bind to the operator 4. RNApolymerase binds to the promoter 5. Transcription of the Lac operon structural genes occurs 6. Ribosomes recognize the ribosome binding site upstream of each of the 3 coding sequences on the mRNA, and translation produces the 3 enzymes *Because an inducer molecule increases its expression, the lac operon is called an inducible operon • absence of lactose means that there are no allolactose inducer molecules to inactivate the repressor; the repressor binds to the operator, reducing transcription of the operon • controls aided by bacterial short lifespan (3 minutes on average) • enzymes also have a short life span and degrade quickly Bio Lecture 10-17 Haffie Time • a typical prokaryotic gene has many types of signals coded in DNA left to right • Green: promotor (2) • Yellow: Sd box • Dark Green: start codon (4) • Blue(which for whatever reason is not on this pic): codons for other amino acids • red: stop codon (1) • Pink: transcription terminator (3) Which of these signals is transcribed but not translated? numbers above for clicker question -1 and 3 • promotors are not transcribed they attract polymerase • start of transcription is downstream of promotor • start codon: is translated and transcribed stop codon is transcribed but there is no tRNAand it doesnt get translated • • termination sequence gets transcribed but not translated • 1 and 3 is the answer What if these signals are changed by mutations? in promotors? -then RNApolymerase could not read the promotor -OR it could increase the promotors efficiency In Sd box? - changes the sequence it might make it more or less functional - effects efficiency of translation In start codon? -destroys start codon and probably kills gene (most likely lethal) In stop codon? Bio Lecture 10-17 -it depends. - can mutate stop codon into another stop codon or... - stop codons are read by translation therefore translation would be effected - ribosome wont stop until they hit next stop codon - evolution has overtime added 2 or 3 stop codons at their ends in order to reduce mutation problems In terminator? -depends if you strengthen or destabilize loop • UGG is the codon for trp • DNA--->MRNA--->POLYPEPTIDE • codons are coded 3’--->5’in DNAbut only understood as 5’--->3’ Substitution mutations may create alternation “silent” codons • • mutations that substitute one codon for another that codes for the same amino acid is called a silent mutation and it does not effect the protein • substitution mutations may create “missense” codons when a codon is changed and forms another amino acid (Thr to pro) • if new amino acid functions like the other protein is not effected much • but if switch an amino acid from positive to negative or hydrophobic to hydrophilic then it would have a dramatic effect • effect depends • substitution mutations may create “nonsense” codons which creates a stop codon that causes premature termination of polypeptide and probably be to short and probably nonfunctional • are usually much more severe • Indel mutations cause a shift in reading frame • during DNAreplication a base pair can be added or deleted via slippage if that happens it shift the reading frame • • if we insert a base into the coding region it disrupts all the pairings down stream • causes a whole new set of amino acids • devastating effects of the function of the protein • What determines the frame a gene is coded in? the start codon says read in threes after me • • start codon sets frame • How many frames are there? How many places are their on a template strand? • 3 possible reading frames on the template strand • Can genes overlap? Could the Indel mutation affect more than one gene at a time? What signals would you find in the trp tRNAgene? (also see paper notes) • they have a promotor and terminator and an anticodon • no codons, they are not translated • no SD box • not involved with ribosomes Bio Lecture 10-17 • tRNAcodes for anticodons that is used during translation • Operons (bacteria) bring genes under the control of one promoter • biochemical pathways are complex so it makes sense to have then under coordinate control • operon is in an inducible state its natural state is off • it is turned on by lactose • lacz and lacy are involved in breaking down lactose • lacI is independent and produces the lac repressor • in absence of lactose, lac repressor binding prevents transcription • lac repressor binds with operon on DNAvia negative(DNA) and positive charges • not base pairing and not covalent bonds Lac repressor is a dimer • • proteins that bind with DNAis usually a dimer • when lac repressor binds with operator it stabilizes the big blue loop which is a loop of double stranded DNA(not a hairpin!!!!) • lac repressor causes this DNAloop which stops polymerase from transcribing the operon (usual state) • most of the time transcription is prevented by lac repressor binding • in presence of lactose, transcription is induced • allows transcription of 3 genes • called a polysestronic message • LACTOSE BINDS ON REPRESSOR! • sestronic=gene • one mRNAcodes for 3 genes lz, ly.la start mRNAcodon isAUG, DNAis TAC • • stop mrna codon: UAG,UGA,UAA PHET LAC OPERON SIMULATION Bio Lecture 10-17 Lecture 14:Eukaryotic Genes (read 13.4, 14.2, 14.3) Although many aspects of eukaryotic gene structure and expression are similar to that in bacteria, the compartmentalization of eukaryotic cells affords dramatically increased opportunities for regulation. 13.4 Review • translation is the assembly of amino acids into polypeptides. Translation occurs on ribosomes. The P,A,E sites of the ribosomes are used for the stepwise addition of amino acids to the polypeptide as directed by the mRNA • Amino acids are brought to the ribosome attached to specific tRNAs.Amino acids are linked to their corresponding tRNAs by aminoacyl-tRNAsynthetases. By matching amino acids with tRNAs , the reactions also provide the ultimate basis for the accuracy of translation • translation proceeds through the stages of initiation, elongation, and termination. In initiation, a ribosome assembles with an mRNAmolecule and an initiator methionine-tRNA. In elongation, amino acids linked to tRNAs are added one at a time to the growing polypeptide chain. In termination, the new polypeptide is released from the ribosome and the ribosomal subunits separate from the mRNA • After they are synthesized on ribosomes, polypeptides are converted into finished form by processing reactions, which include removal of one or more amino acids from the protein chains, addition of organic groups, and folding guided by chaperons. • proteins are distributed in cells by means of signals spelled out by amino acid sequences at the N-terminal end of the newly translated polypeptide. • Mutations in the DNAtemplate alter the mRNAand can lead to changes in the amino acid sequence of the encoded polypeptide.Amissense mutation changes one codon to one that specifies a different amino acid, a nonsense mutation changes a codon to a stop codon, and a silent mutation changes one codon to another codon that specifies the same amino acid.Abase- pair insertion or deletion is a frameshift mutation that alters the reading frame beyond the point of the mutation, leading to a different amino acid sequence from then on in the polypeptide. 14.2: Regulation of Transcription in Eukaryotes • Operons are not found in eukaryotes. Instead, genes that encode proteins with related functions are typically scattered through the genome, while being regulated in a coordinated manner two general types of gene regulation occur in eukaryotes. Shortterm regulated involves • relatively rapid changes in gene expression in response to changes in environmental or physiological conditions. Long-term regulation involves changes in gene expression that are associated with the development and differentiation of an organism Bio Lecture 10-17 • gene expression in eukaryotes is regulated at the transcriptional level (where most regulation occurs) and at posttranslational levels • regulation of transcription initiation involves proteins binding to a gene’s promoter and regulatory sites.At the promoter, general transcription factors bind and recruit RNA polymerase 2, giving a very low level of transcription.Activator proteins bind to promoter proximal elements and increase the rate of transcription. Other actiavtors bind the enhancer and, through interaction with a coactivator, which also binds to the proteins at the promoter, greatly stimulate the rate of transcription. • The overall control of transcription of a gene depends on the particular regulatory proteins that bind to promoter proximal elements and enhancers. The regulatory proteins are cell-type specific and may be activators or repressors. This gene regulation is achieved by a relatively low number of regulatory proteins, acting in various combinations. • The coordinate expression of genes with related functions is achieved by each of the related genes. The change in chromatin structure than trnscriptionally inactive genes. The change in chromatin structure that accompanies the activation of transcription of a gene involves chromatin remodelling-specific histone modifications- particularly in the region of a gene’s promoter • Sections of chromosomes or whole chromosomes can be inactivated by DNAmethylation, a phenmenon called silencing. DNAmethylation is also involved in genomic imprinting, in which transcription of either the inherited maternal or the inherited paternal allele of a gene is inhibited permanently 14.3 Posttranscriptional, Translational, and Posttranslational Regulation • Posttranscriptional, translational, and posttranslational controls operate primarily to regulate the quantities of proteins synthesized in cells • Posttranscriptional controls regulate pre-mRNAprocessing, mRNAavailability for translation, and the rate at which mRNAs are degraded. In alternative splicing, different mRNAs are derived from the same pre-mRNA. In another process, small single-stranded RNAs complexed with proteins bind to mRNAs that have complementary sequences, and either the mRNAis cleaved or translation is blocked • Translational regulation controls the rate at which mRNAs are used by ribosomes in protein synthesis • Posttranslational controls regulate the availability of functional proteins. Mechanisms of regulation include the alteration of protein activity by chemical modification, protein activation by processing of inactive precursors, and affecting the rate of degradation of a protein. Haffie Time • nucleus and compartmentalization enables ribosomes to stay away from the mRNAinitially • The difference is the jobs they have. mRNAstands for messenger RNA, it takes the information of the DNAfrom the nucleus to the ribosomes. tRNAstands for transfer RNA, it adds one link to a growing polypeptide chain during translation. rRNAstands for ribosomal Bio Lecture 10-17 RNA, it is the main component of ribosomes, and plays a large part in the creation of new proteins. I hope this helps! The synthesis and function of a typical tRNAmolecule requires complementary base-pairing with 1. itself 2. DNA 3. other RNA 4. one particular amino acid • 1,2, and 3 • all RNAs come from transcription • trna comes from transcription (WHEN THEYARE MADE)and during that transcription they are base paired with DNA • amino acids are not bases and therefore cannot pair with anything How many stop codons (in DNA) are in this picture? (see Lecture) • 4 genes in lac operon. Lacz, lacy, lacA, lacI. • 4 genes need 4 stop codons • Red: transcriptional regulation regulation of transcription initiation • • determines which genes are described • Green: posttranscriptional regulation • Determines types and availability of mRNAs to ribosomes Bio Lecture 10-17 • red-brown: translational regulation • determines rate at which proteins are made • Posttranslational regulation • determines availability of finished proteins • Nuclear gene structure is more complicated than organelles genes dont have to have introns but they often do and have many sometimes (100) • • proximal regions are protein binding sites near promoter (usually upstream) • enhancers can be quite far away from the gene they regulate • RNApolymerase 2 does not bind “naked” promoters • bacteria only one polymerase • nucleus there are several and RNApoly 2 is recognized as the major one • poly 2 only find promoters attractive only if proteins are already binded to TATAsite, TATA protein • transcription begins downstream of promoter • Transcription factors regulate efficiency of polymerase binding • TATAbinding proteins attracts poly 2 and other transcription cofactors that bind to the DNA How do proteins bind to DNA? • Electrostatically DNAbackbone is negatively charge and proteins can be positively charged • • only works if proteins have particularly shapes to fit into helix • helix turn helix DNAbinding • BLUE is string of amino acids • lac operon was a helix turn helix • Zinc finger DNAbinding (binds the major groove) • amino acids that can associate with zinc ions (zinc in protein) and form a particular shape that recognizes the DNAsequence • amino acids wrap around zinc ion to form specific DNAbinding protein Bio Lecture 10-17 • • Leucine zippers hold 2 monomeric DNAbinding proteins together • DNAbinding proteins sometimes act as dimers the zipper keeps the two dimers together • • once proteins bind with TATAand the polymerase binds to the promoter the DNAfolds over to contain it • need activators at enhancer and promoter • proteins also bind with enhancer • the enhancer is what folds the DNAand stabilizes the whole complex • the enhancer does not have to be in one spot, not so position dependent • it is position independent • promoter must be just upstream of gene • if enhancer is moved then gene won’t transcribe as well What if a virus come with a megaenhancer? Maybe the gene will be over expressed Bio Lecture 10-17 • took promoter and invert it • it would send transcription the other way • promoters matter which way they are facing • enhancers don’t • they can fold or loop and fold they are not directional or position dependent • • enhancer holds proteins and activators onto the promoter region to make it attractive to poly 2 • Unique combinations of activators control specific genes • the genes in the lens of your eye need to be expressed differently than those in your liver • one way to do that is to make tissue specific activator proteins • one way to express genes differently is by regulating their enhancers or the attractiveness of the promoters • the most efficient way to regulate expression is at transcription • 5’end there is a G cap (phosphorous cap) • other end is the polydenylation signal • this is how transcript