AP Bio unit 6 gene expression if you're getting ready for the AP Bio exam or for a comprehensive unit 6 test and you're looking at a diagram like this which shows production of RNA modification of RNA into mRNA and then translation of mRNA into protein and you're thinking this is complex stuff then don't worry that's completely reasonable it is complex stuff and what this video is going to do is help you get ready for both the AP Bio exam and that unit 6 test here's what this video is going to cover DNA and RNA structure and function DNA replication transcription translation and the genetic code regulation of gene expression in procaryotes which is about operons eukaryotic gene expression mutation and horizontal Gene transfer and biotechnology I'm Glenn Wilkenfeld also known as Mr W I'm a retired AP biologist teacher and I love teaching b i o l o g y when I taught AP Bio most of my students got fours and fives on the AP Bio exam and this video is designed to help you do the same one of my keys to success as an AP Bio teacher was my use of the interactive AP biology curriculum learn biology.com which I wrote for my own students to help them succeed I'm anxious to share it with you please sign up at learnbiology dcom biology topic 6.1 DNA and RNA structure here are some of the questions that we'll be addressing describe the structure and function of DNA and RNA compare contrast how procaryotic and eukaryotic DNA is organized what is a plasmid describe the structure of DNA DNA is a double stranded helical molecule composed of nucleotide monomers in this flattened out representation of DNA here's one strand here's the other strand in this helical representation you can see one strand another strand because there's two strands it's a double helix the monomers are nucleotides they consist of a five carbon sugar called deoxy ribos hence deoxy ribonucleic acid a phosphate group and one of four nitrogenous bases so it doesn't have to have this exact structure it can vary over here in terms of the nitrogenous base each strand consists of coal bonded deoxy ribos sugars and phosphate groups which comprise DNA sugar phosphate backbone within the Helix bases with complimentary shapes bind through hydrogen bonds thyine is complimentary to adenine guanine is complimentary to cytosine in the case of adenine and thyine you can see the hydrogen bonds that form between the oxygen over here the hydrogen over here hydrogen over here nitrogen over here the bonding follows base pairing rules that you have to commit to memory adenine binds only with thyine a binds with t guanine binds only with cytosine G binds with C for the nucleotides to bind they have to be oriented upside down relative to one another making the two strands anti-parallel this strand has its five Prime end over here it's three prime end down here and this strand is the opposite anti-parallel like this explain how DNA's structure allows it to serve as the molecule of heredity we'll start with information storage the four bases can occur in any order I've represented them over here is a g over and over again to show the structure but there could be three A's in a row followed by two C's followed by a t followed by whatever the sequence isn't determined by DNA's chemistry that allows the sequence to be an informational code that specifies sequences of RNA and protein replicability the specific base pairing a GC that we talked about previously allows each strand to serve as a template for the synthesis of a complimentary strand during DNA replication which we'll talk about in a moment that also ensures High Fidelity transmission of genetic information from parent cells to daughter water cells DNA is highly stable its double helical structure protects the sequence of bases that are inside but while it's stable it's also capable of mutation mutability is the fourth characteristic there's a low level of mutation where bases can change from one to another either spontaneously or caused by mutation causing factors in the environment and that allows for change in this code which allows for evolution compare and contrast the functions of DNA and RNA DNA is the molecule of heredity in all organisms anything that is cell-based life which is all life has DNA as its molecule of heredity as the stuff that genes are made of RNA is the hereditary molecule in some but not all viruses viruses that you might know about that are RNA based include HIV and SARS a form of which just caused the covid-19 pandemic in all organisms RNA is involved in information transfer related to protein synthesis how DNA becomes RNA and how RNA becomes proteins and that includes forms of RNA such as mRNA TRNA and rrna in ukar RNA is also involved in the regulation of gene expression and this previews some topics that we'll talk about later in this unit that includes splicing out introns non-coding DNA from pre-mrna to create mRNA and regulating protein synthesis compare and contrast how genetic information is stored in procaryotes and ukar procaryotes store their DNA in looped circular chromosomes in other words the beginning and the end is connected it's sometimes ascribed as circular but looped is really more accurate the genomes of bacteria and ARA range from about a 100,000 base pairs to 10 million base pairs and their DNA is naked it's not wrapped around a protein scaffold in ukar Nots the DNA is organized into multiple linear chromosomes so there's one end and there's another end and the DNA is wrapped around these proteins that are called histones you carry itic genomes are much larger than procaryotic genomes the human genome is one example consists of 3.2 billion base pairs but there are some plant genomes that consist of 150 billion base pairs what are plasmids what's their function how are they used in genetic engineering plasmas are small extra chromosomal Loops of DNA commonly found in bacteria less commonly in archa rarely in UK carots here's the main bacterial chromosome these Loops also made of DNA are the plasmas they're involved in horizontal Gene transfer between bacterial cells through a process called conjugation these transfer genes because they're transferring DNA that codes for protein from one cell to another they're often for antibiotic resistance plasmas are commonly used in genetic engineering as a vector for replicating DNA and for expressing engine genes within bacterial cells both horizontal Gene transfer and genetic engineering are going to be covered later in unit 6 topic 6.2 DNA replication some of the questions we'll be addressing why is DNA replication described as semiconservative what are the key enzymes involved in DNA replication and what do they do what's the difference between DNA replication at the leading and lagging strands what are okazaki fragments on a big picture level describe how DNA replication occurs and what the term semiconservative means during DNA replication a team of enzymes using each strand of the double helix as a template synthesizes New Daughter strand here's the original strand enzymes pull that strand apart and that results in two daughter strands that are each single strands these single strands serve as as a template and what that means is that nucleotides that are available in the nucleus following the base pairing rules will bind with the exposed strand a will bind with t c will bind with G Etc the result is that each daughter DNA double helix consists of one conserved strand from the parent molecule and another strand that was synthesized a new in these daughter molecules one of the strands is is this strand here we go the other strand is this strand whereas these strands over here are new semiconservative one strand is conserved the other strand is new you can see that represented here with a kind of color coding where the parent strand has both strands colored red and you can see in the daughter strands one strand is red conserved one strand is orange new that method of replication is known as semiconservative replication describe how DNA replication starts in the model of DNA replication that we're about to talk about there's a lot of simplification compared to how the process actually works in nature but don't worry this is exactly what you need to know for AP biology the process begins when an enzyme called helicase over here at B finds a sequence called the origin of replication that basically says start replicating in here and separates the double stranded DNA as you know that means breaking the hydrogen bonds that are holding the two strands together this exposes two single strands and it creates a structure that's called a replication fork describe the roles of DNA polymerase primase and primers in replication let's get oriented here before we start this is a replication fork this is DNA helicase that's opening up the parent strand exposing the nucleotides in the two daughter strands DNA polymerase is this enzyme shown over here and over here it's the key enzyme involved in creating new DNA the parent DNA is shown in dark blue the new DNA that's coming in is represented in light blue the nucleotides bind based on base pairing rules DNA polymer doesn't know which nucleotide should fit the knowledge is basically in the template strand so if there's a c over here then a g will bind if there's an a over here then a t will bind what DNA polymerase does is it binds new nucleotides to the three prime end of a growing Strand and that's a sugar phosphate Bond DNA polymerase has a limitation it can only add nucleotides to an existing strand so think of it as an enzyme and it hasn't active site its substrate is the pre-existing Strand and the new nucleotide that came in so to start the process DNA polymerase needs an RNA primer a couple of bases of RNA that DNA polymerase can start connecting DNA nucleotides to here's the primer shown over here here it's shown and it's represented by number four there's another enzyme that can come to an open replication fork and start laying down that primer and that's represented here at five it's called primase primase lays down the primer what role does single strand binding proteins play during replication the single strand binding proteins are shown at eight and what they do is they keep the double helix from rewinding so that all of these other enzymes can get into place and Carry Out replication how is DNA replication at the leading strand different from replication at the lagging strand in each replication fork there's going to be a leading Strand and a lagging strand in the leading strand which is shown over here at J DNA replication is relatively continuous because DNA polymerase at G is following the opening replication fork that's being created by helicase over here in the lagging strand which is shown at L DNA polymerase synthesizes in the opposite direction from the opening replication fork so what you have to imagine is that DNA helicase opens up the Helix a little bit DNA polymerase gets in and starts synthesizing and then it opens up a little bit more well DNA polymerase can't go in this direction it can only go in the 5 to3 Direction so that means another DNA polymerase com comes in and synthesizes this over here and yet another and each time there's a primer what that means is that on the lagging strand DNA replication is discontinuous and it's built from short sequences that are called okasaki fragments named after the researchers who discovered that this is how the process works describe the roles of DNA polymerase 1 and ligase during DNA replication DNA polymerase 1 shown over here at K removes the RNA primers here's one over here here's a whole bunch over here and it replaces the RNA with DNA another enzyme called DNA liase is required to finish the process and create the complete daughter strands what it does is it seals the gaps between fragments with sugar phosphate bonds topic 6.3 transcription here are some of the questions that we'll be addressing explain the overall flow of genetic information within cells what are the principal forms of RNA and what is the function of each explain what happens during transcription explain the overall flow of genetic information within cells this is the central dogma of molecular genetics which is DNA makes RNA makes protein information flows from a sequence of DNA triplets to a sequence of mRNA codons to a sequence of amino acids what is a gene if you've been following this series we looked at this slide in unit five but now let's look at it again in the context of molecular genetics a gene is the basic unit of heredity passed from parent Offspring it determines a trait in terms of molecular genetics it's a sequence of DNA nucleotides that codes for RNA which codes for protein list the principal forms of RNA and describe the function of each one mRNA or messenger RNA is a linear molecule and it brings instructions from DNA to ribosomes R RNA ribosomal RNA makes up the catalytic part of ribosomes and binds amino acids together during protein synthesis ribosomes are these particles that are composed of our RNA and protein we look at them in depth later but they're essentially enzymes and they're the enzymes that bind amino acids together during protein synthesis trnas Transfer RNA bring specific amino acid to the ribosomes again for protein synthesis small rnas are a large group of rnas of different shapes and sizes and they're involved in eukaryotic Gene regulation what happens during transcription transcription is the creation of RNA which we see over here in Blue from DNA every Gene begins with a promoter region that indicates that that's where the gene starts and during transcription an enzyme called RNA polymerase binds with a promoter on DNA then it transcribes the sequence of DNA bases on DNA's template strand into a sequence of RNA RNA polymerase like all of the enzymes involved in working with DNA reads the DNA in the thre Prime to five Prime Direction and synthesizes new RNA in the five Prime to three prime Direction and when the RNA polymerase reaches a Terminator region which is at the end of the gene it dissociates from the DNA ending transcription Define and describe template strand minus strand non-coding strand or anti-sense strand in relationship to RNA transcription the template strand it's this one over here in blue it's also called The noncoding Strand The antient Strand and the minus strand that's what gets transcribed from DNA into RNA the complementary strand to the template strand is called the coding strand why because you can see that it has the same sequence of nucleotides as the MRNA will here's the coding strand G GT T AA here's the RNA that's being produced g g uu U substitutes for T in RNA AA so GG uua a g g TTA AA it's the same why is it the same because it was created in response to this template strand over here that's why the coding strand is called the sense strand or the positive strand what are some unique features of procaryotic transcription procaryotes don't have a nucleus there's no separation between the genetic material and the cytoplasm as a result in procaryotes transcribed RNA which is shown here at D can immediately be translated by ribosomes into protein and that's what you see as the these strands over here often multiple ribosomes read the same RNA strand these multiple ribosomes are sometimes called polysomes and you can see them in a more zoomed in version over here the genetic code and translation SL protein synthesis here are some of the questions that we'll be addressing what is the genetic code and how does it work what are the key molecules and structures involved in protein synthesis describe the process of protein synthesis what is the genetic code how do you read a genetic code chart use the code to translate a GU a a guu into protein the genetic code is the code used by living things to translate nucleotide sequences into amino acid sequences we've talked about how DNA makes RNA makes protein DNA to RNA is transcription now we're going to get from RNA to protein in the genetic code groups of three RNA nucleotides so like for example Aug gu they're called a codon and they code for one amino acid codon code one the code is nearly Universal nearly every living thing uses it in exactly this way there are some minor exceptions it's specific every codon can determine one amino acid but it's redundant there are synonymous codons Aug gu a a guu codes for methionine veine lysine veine how did I do that here's how you use the code a u this is the first nucleotide in the RNA codon this is the second nucleotide in the RNA Cod on and this is the third so you work from the inside out a u g codes from methionine g u c codes for veiling a a g codes for lysine and g u u codes for veiling so we can see how gu and guu are synonymous and that's an important relationship often the first two nucleotides are more important than the third one and ones that have the same two first nucleotides are often synonymous use the genetic code dictionary below to code out cuc G Au GCA gu C cgu the code that we looked at on the previous slide is a circular code I actually think it's easier to use though you'll frequently see codes like this that are tabular in this code the first base is represented on this column the second nucleotide base is represented over here and the third is going to be one of these four let's demonstrate how that's used c c is the first code on c u and here's C that codes for Lucine G Au G is the first base a is the second second base U codes for asparagine GCA g c a codes for alanine guu g u c codes for veine and cgu C G codes for arginine here you see all of the amino acids and their corresponding codons represented here what what's the big picture of translation who are the key players mRNA is shown over here at G2 it contains the codons this group over here Aug or this auug or this ccg that specify the order of amino acids here are amino acids shown over here the ribosome connects amino acids to create a polypeptide ribosome is represented this K and what it does is it'll connect for example this amino acid Proline to this growing chain of amino acids a chain of amino acids is also referred to as a polypeptide remember that's the first level of protein structure the linear sequence of amino acids trnas are shown over here at letter O here's a TRNA and trnas bring amino acids so such as this one over here to this ribosome mRNA complex trnas have an anti-codon such as this over here GGG G letter H and an amino acid binding site over here in the next slides we'll put the entire process together step by step what is the role of the ribosome in Translation what are the key parts of ribosomes to know ribosomes are General purpose protein factories they can take any mRNA which is what brings it information and convert it into any sequence of amino acids they're the enzymes that string together amino acids to form polypeptides and they do that following the instructions in mRNA they have a large and a small subunit and they have three TRNA binding sites the E is the exit site that's where the TRNA that's given up its amino acid will leave from the pite holds the polypeptide and the new amino acid comes into the aside describe how translation protein synthesis begins processed mRNA that means mRNA that's ready to be translated that doesn't have any introns in it leaves the nucleus it'll leave through a nuclear pore the small ribosomal subunit will bind with the MRNA and make its way over to the start codeon which is a ug that's where translation begins that small subunit then Waits quote unquote for a TRNA with a matching anti-codon to bind with the Starcon so this TRNA has the anti- code on UAC which complements Aug this first TRNA is carrying the first amino acid methionine then the large subunit binds with the small subunit the ribosome is now complete and that first TRNA with methionine is located in the ribosome's psite that's the middle binding site describe the elongation phase of translation the next TRNA comes to the a site and it Bears a new amino acid here it is the ribosome then catalyzes a peptide bond between the p and asite amino acids so here's a peptide bond that's forming between methionine and veine then the ribosome translocates it moves over one more codon so that means that a dipeptide is now hanging off the pide amino acid the aside is empty and there's a TRNA in the eite but it's not connected to the polypeptide it's not connected to any amino acid now what happens is that the TRNA that's in the east side exits the new TRNA enters at the asite the as's empty over here a new TRNA comes in the ribosome is going to catalyze a bond between that new amino acid and the existing dipeptide so there's going to be a tripeptide that's temporarily at the aite but the process is going to continue will'll be another round of translocation followed by exit followed by another TRNA that's charged with an amino acid coming in followed by another peptide bond happening and that continues along the entire length of the MRNA describe the termination phase of translation the ribosome gets to a stop codon the stop codons in the genetic code have no corresponding TRNA instead what they do is they code for a release Factor That's A protein that can bind with the stop codon and induce certain changes in the MRNA TRNA ribosome complex and those changes cause the ribosome to dissociate and the polypeptide to be released the only thing that needs to happen now is for this polypeptide to fold up into a functional protein translation is done at learnes biology.com we understand why students struggle with AP Bio it's a hard course but we have a plan for your success go to learn down biology.com sign up for a free trial and complete our interactive tutorials and interactive AP Bio exam reviews we guarantee you a four or a five on the AP Bio exam see you on learn biology.com topic 6.5 to 6.6 regulation of gene expression part one operons here are some of the questions we'll be addressing what are operons what's the difference between an inducible and a rep pressible operon how does the trip operon work explain how the Lac operon works let's start with a little context eoli is a bacterial cell that lives in our colons that coli is related to colon and it also lives in the colons of many other animals the colon is the large intestine eoli has about 4,000 genes this is a chromosome map of eiz chromosome and it shows a small portion of these 4,000 genes which code for a variety of proteins the overall Genome of eoli consists of about 4 million base pairs a t C's and G's and this leads to a question of Regulation which is what's the control system for turning its genes on and off let's start by responding to this very general question what is is an operon one definition is that an operon is a cluster of genes transcribed as a single RNA here we have a portion of DNA that's labeled structural genes and it's all transcribed as one RNA transcript but then that RNA is processed so that it's producing a variety of enzymes a cluster of genes transcribed as a single RNA but our Focus in AP biology is that an operon is a mostly procaryotic system of Gene regulation that has control elements that allow for Gene regulation describe the structure of an operon here we see a string of DNA and it's an operon it consists of structural genes and those are genes that code for protein there's an operator which is where a repressor protein binds and that enables this system to be regulated there's a promoter where RNA polymerase binds and there's a regulatory Gene that produces the regulatory protein that regulatory protein is generally A repressor protein that binds at the operator how does the trip operon work the trip operon is a system that codes for a series of enzymes that make tryptophan that's what the structural genes do these enzymes work as part of a metabolic pathway that codes for tryptophan which is one of the 20 amino acids but it's also a regulatory system that only turns on production of these enzymes at certain moments if there's no tryptophan in the environment then the regulatory protein over here which is produced by the regulatory Gene and that's pretty much always on that can't bind with the operator look at the shape of this regulatory protein over here and notice that this part won't bind with this and really what we're talking about is a protein with a complex shape that can actually bind with a sequence of DNA because that's what the operator is it's DNA that means that RNA polymerase can bind at the promoter and it can roll down the length of the Gene and transcribe the structural genes creating these enzymes when tryptophan is in eiz environment that tryptophan the amino acid will diffuse into the cell what will happen when tryptophan is in the cell's environment then what'll happen is it'll bind with the repressor protein and what it does that will cause the repressor protein to change shape think of this like an enzyme that's doing an aleric shift binding over here causes a chain over here how and why this is a protein that has Alpha helices and pleated sheets and it's very Dynamic so The Binding over here causes a change over here that enables this regulatory protein a repressor to bind with the operator when it does it blocks RNA polymerase that means that RNA polymerase can no longer transcribe these structural genes to make enzy s that synthesize tryptophan that makes a lot of sense the basic rule is if tryptophan is present don't make it it's an adaptation for saving energy trip is therefore called a repressible operon and tryptophan is the co- repressor this protein when it binds with tryptophan blocks the operator repressing the system transcription becomes impossible that's the trip operon how does the Lac operon work we just looked at the trip operon which controls the synthesis of enzymes for synthesizing tryptophan what about this Lac operon the Lac operon is an inducible operon as opposed to trip which was a repressible one and it codes for enzymes that digest lactose a disaccharide so here's lactose you can see it's composed of two sugar monomers and the enzymes that digest lactose will break it down into glucose and galactose what happens when lactose is in the environment remember these bacteria live inside our guts so if you had ecoli in your guts and you drank a glass of milk the sugar in the milk which is lactose would then be in the environment of eoli that lactose will diffuse into eoli once lactose is inside eoli it binds with the repressor protein here's lactose it's binding with the repressor protein and notice the effect in this case the lactose causes the repressor protein to change shape so it can't bind with the operator that keeps the operator free and when RNA polymerase binds at the promoter it can roll down the length of the operon it can transcribe the structural gene gen and those structural genes produce enzymes that break down lactose into monosaccharides and also increase the permeability of ecoli cell membrane so that more lactose can enter when lactose is absent however there's no lactose available to bind with the repressor the repressor is default shape lets it bind with the operator RNA polymerase therefore after binding with a promoter can transcribe the structural genes the rule is if lactose is absent don't make genes to digest it again think of this as a metabolic adaptation this saves energy don't make enzymes to digest something when the thing that you're digesting isn't around lack is therefore an inducible operon it can be induced to be turned on what turns it on lactose lactose is the inducer the Lac operon is a negative feedback system explain think about how the Lac operon works lactose turns the system on turning the system on removes lactose from the system why because turning the system on allowing RNA polymerase to transcribe these genes allows for the production of enzymes and proteins that enhance lactose digestion that enhance lactose digestion will make all of this lactose go away when all of this lactose goes away there'll be no more lactose to bind with the repressor which will then bind with the operator the result is that the system turns off and that's negative feedback where the output of the system has the effect of quieting or repressing the system you can say the same thing about the trip operon it's also a negative feedback system why the absence of tryptophan starts transcription when tryptophan is not in the environment then the regulatory protein can't bind with the operator that enables RNA polymerase to transcribe these structural genes producing these enzymes that are part of the metabolic Pathway to produce tryptophan that produces tryptophan and the production of tryptophan puts tryptophan at high enough concentration in the cell so that it binds with the repressor protein changing its shape allowing the oppressor protein to bind at the operator shutting down transcription that turns the system off that's also negative feedback both trip and lack negative feedback systems even though lack is an inducible system and trip is a repressible system the graph below shows the growth of an ecoli culture that's fed with both glucose and lactose X and Y show the glucose and lactose concentrations so note how the glucose concentration is going down over here and they're to set the lactose concentration maintains itself and then goes down over here the red line shows the growth of the bacteria over 9 hours there are two lags in growth one is at B over here between a and c and the other one is here at D what's happening eoli prefers to metabolize glucose why because glucose is a monosaccharide lactose is a disaccharide and as you know glucose is the fu that goes right into the glycolysis process that begins cellular respiration now up to point a eoli eats glucose and grows rapidly but then the glucose starts to run out as the glucose starts to run out there's a lag in growth during activation of the Lac operon and lactose digesting enzymes from C to D the Lac operon is churning out those enzymes that break down lactose into glucose and galactose but at a certain point lactose runs out and then there's another lag which might be a permanent lag until another food source is introduced into the culture the key idea is that glucose is easier to digest than lactose glucose will be metabolized it'll be digested first followed by lactose there was a graph like this that was an frq on one of the previous tests and this is why you have to understand operons in order to succeed in AP biology regulation of gene part two eukaryotic Gene regulation here are some of the questions we'll be addressing what are acetalation and methylation and why is eukaryotic Gene regulation so complex what is premrna describe some of the posttranscriptional modification that has to happen to premrna and ukari before it can be translated into protein explain how the organization of eukaryotic genetic material into introns and exons can increase phenotypic variation Gene regulation in multicellular ukar key issues organisms like you and me and lizards and redwood trees and Jellyfish any multicellular UK carote is composed of trillions of cells organized into specialized tissues we have 46 chromosomes three billion base pairs in each hloy genome and 20,000 genes Gene regulation is a big and complex issue here are some more parts of that issue every single cell has the same DNA but cells need to know which genes to express as they develop and Gene regulation as we just saw with procaryotes and operons is also influenced by factors in the environment how do genes get turned on and off note that most eukaryotic DNA is noncoding so what's the difference between the coding DNA and the non-coding DNA and genes contain introns we've mentioned these before now we'll really look at them in depth in eukariotic cells what determines which genes are expressed let's start with this fact in any cell in a multicellular organism most of the DNA is not not expressed you have cells that make up the lens of your eye those cells Express a single protein that means that 19,900 something other proteins are not being expressed all those genes are turned off those genes that are turned off are tightly packaged around proteins that are called histones that's what these diss over here represent there's an additional chemical modification which is called methylation it's the addition of a methyl group that carbon attached to three hydrogens and that prevents transcription in the few genes within any cell that are turned on there's a process called acetalation that loosens up the DNA and that makes it possible for RNA polymerase to come in find the promoter and transcribe the genes what is epigenetics we just talked about how in most cells most of the DNA is not transcribed it's silenced it's turned off only a small number of genes are turned on what's the difference that is all defined by this newly emerging topic that's called epigenetics epigenetics are changes in DNA expression that evolve reversible chemical modifications of DNA or modifications in DNA packaging chemical modifications of DNA methylation modification in DNA packaging wrapping around these proteins that are called histones but the genes themselves the sequence of nucleotides is not changed it's a level above the genetic level that's why it's called epigenetics it's responsible for the differentiation of tissues during development why are skin cells expressing skin proteins where fingernail cells are expressing fingernail proteins and muscle muscle cells expressing muscle proteins those are all about epigenetics because all of those cells contain the same genes somewhat astonishingly sometimes these changes can be transmitted from one generation to the next that's a newly emerging field of study and that's intergenerational transmission of epigenetic modifications of the genome what's the connection between epigenetics cell differentiation and gene expression the key idea one that needs to be memorized is that all cells in the same organism are genomically equivalent every cell in your body except for your gametes has the same DNA all cells are descended from the zygo that's shown at number one in this diagram all cells have the same DNA that's shown at three and four cells differentiate because they express different genes and that relates to the epigenetic modifications that we just talked about in the previous slide describe on a big picture level how transcription is regulated in ukar and eukariotic cells previously we talked about operons which is how genes can be turned on and off in response to environmental changes ukar have to be able to do that too some of that relates to acetalation methylation histones the things we've talked about but some of this is on a more immediate regulatory level so let's look at the regulatory processes that occur in ukar ukar possess regulatory DNA sequences that interact with regulatory proteins to control transcription I know that this diagram looks horrifyingly complex but you really only need to know it on a basic level so you can understand questions that might come your way on unit test or on the AP Bio exam some of these regulatory sequences include promoters we've talked about those in the context of transcription so there are promoters shown at letter e there are also enhancers that are shown at letter a and what they do is they increase the probability that a gene will be transcribed they enhance that possibility interactions between activator proteins they're shown at B DNA bending proteins that's at F mediator proteins at G and general transcription factors H enable RNA polymerase shown at letter i to bind making transcription possible all you really need to know is that this kind of system is one that's used for eukariotic Gene regulation you'd never be asked to differentiate between these mediator proteins at G and these General transcription factors you just just need to know the Big Picture This Is What eukariotic gene regulation can look like how can gene expression be coordinated in different body tissues during development as we've discussed different tissues Express different genes but those different tissues can also share common regulatory sequences that enable the transcription of genes within those various tissues to be coordinated an example of that is that the tissue in a male Lion's neck skin talking about this over here and their muscle tissue Express different genes one's expressing the hair that makes up the Mane and the other is expressing the tissue in the muscle but both of those tissues share a common testosterone receptor Gene that testosterone receptor Gene gets expressed as a cytoplasmic receptor and therefore when test testosterone gets released into the body it binds with the testosterone receptor this becomes a transcription factor that goes into the nucleus and activates genes the genes that are activated are going to be different depending on whether those cells are in the Lion's neck or in the Lion's muscle tissue but that leads a single hormone in this case to be able to induce changes in different tissues it's coordination of gene expression in different body tissues what are introns exons what's required to make translatable mRNA in UK carots introns are intervening sequences of DNA within genes they're transcribed into premrna here's an intron in DNA here's an intron in pre-mrna exons are DNA that becomes RNA that ultimately becomes mRNA that gets expressed into protein it gets translated into protein and that is just a bit of the processing of premrna that has to happen in ukar here's the process of transcription relatively straightforward but then what we need to have happened is all of these introns need to be cut out and then the MRNA needs some mod ation so that it can survive in the cytoplasm and be translated by a ribosome into protein we'll see that in a couple of slides describe some of the post-transcriptional modification that has to happen to pre-mrna in ukar before it can be translated into protein in eukariotic cells premrna is what's transcribed from a protein coding Gene so this is DNA over here you can tell because it's a double helix and this is premrna right over here at number number two before it can be translated into protein that pre-mrna has to be processed in several ways it has to get an addition at its five Prime end of a GTP cap and a thre Prime poly a tail poly a tail it just means it's adenine adenine adenine adenine all over again as we've discussed previously introns those intervening sequences that don't code for protein need to be excised they need to be cut out and then the fragments that consist of the exons need to be spliced together then you wind up with mRNA that can be translated into protein what is the function of the five Prime GTP cap and the thre Prime polyat tale that's added to mRNA during eukariotic RNA processing that five Prime GTP cap which is shown over here at G protects the MRNA from breakdown by enzymes in the cytoplasm and it also assists the MRNA in leaving the nucleus and binding with a ribosome the three prime polyat tale shown over here that makes the MRNA more stable and it delays its enzymatic breakdown by enzymes that are in the cytoplasm explain how the organization of eukaryotic genetic material into introns and exons can increase phenotypic variation as we've discussed before exons are expressed sequences they're translated into amino acid sequences introns are intervening sequences that are spliced out of mRNA before translation here we have DNA and here we have premrna and what we've got to do is we've got to cut out these introns but in ukar there's a process called alternative splicing through alternative splicing exons can be spliced together in alternative ways allowing for the production of multiple protein versions from the same premrna transcript so for example in this mRNA and in this protein what we've done is we've dropped out a couple of the exons there's Exxon one three four and six here's another version of the same protein Exxon 1 2 five and six and here's yet another one Exxon 1 2 4 five and six the basic idea is that each of these exons codes for what's called a functional domain a piece of the protein that can actually do something you put those functional domains together and you get proteins with slightly different functions they're all within the same close family they're all from the same gene but they're different manifestations of those genes and they provide for additional phenotypic variation that's found in ukar but it doesn't happen in procaryotes who don't have the intron Exon organization of their coding genes explain the role of small rnas in eukaryotic Gene regulation small rnas are exactly what they sound like they're segments of RNA that don't consist of a huge number of nucleotides yet they play important regulatory roles in the cell one of these is micro rnas micr rnas are particularly small and they play a role in what's called posttranscriptional control of gene expression that's exactly what it sounds like it's after transcription a key process that micrornas are involved in is called RNA silencing here's how it works here's DNA that DNA will contain a gene that codes for a micro RNA not all genes code for proteins some of them just code for rnas in the same way as pre-mrna needs to be processed before it matures there is processing of the Prem microrna to make it into mature microrna in the same way as ribosomal RNA will connect with protein this micro RNA will connect with a protein that's called an RNA silencing complex protein together that RNA plus the RNA silencing complex protein will do one of two things if the microrna completely matches 100% part of a sequence within an mRNA then that complex will cause the MRNA to be degraded and destroyed if on the other hand there's a partial match then this complex will cause a pause in Translation in either case what have we done we've changed expression of a gene through microrna are you asking yourself how am I going to get a four or a five on the AP Bio exam it's a good question because it's a hard test but we have a plan for your success go to learn biology.com sign up for a free trial and complete our interactive tutorials and interactive AP Bio exam reviews we guarantee you a four or five on the AP Bio exam see you on learn Das biology.com topic 6.7 part one mutation here are some of the questions that we'll be addressing what is a mutation what's a point mutation distinguish between silent mutations nonsense mutations and missense mutations mutations can be positive negative or neutral explain what is a mutation what's a point mutation a mutation is a random change in DNA or an entire chromosome a point mutation is a change in a single nucleotide you see that here with the nucleotide C mutating to the nucleotide T distinguish between silent mutations nonsense mutations and missense mutations silent mutations are are mutations that result in the same amino acid being coded for the DNA changes but the amino acid and the protein doesn't why because the genetic code is redundant with many codons coding for the same amino acid a nonsense mutation is a mutation that inserts a stop codon instead of an amino acid and a missense mutation changes the amino acid from one to another we see a silent mutation over here the original DNA is lysine the DNA that's now being coded for despite this mutation is lysine here's a nonsense mutation where instead of Lysine we have a stop codon and here we have missense mutations one is coding for arginine instead of Lysine and one is coding for threonine the effect of a missense mutation depends on the chemistry of the substitution this isn't a term that you need to know but a conservative missense mutation is one where the chemistry is of less significance so lysine is a basic amino acid it has this amino group over here at the end and so does Arginine so that might not change the structure of the protein very much it might not change it in a functional way that is really observable in a phenotype it's not clear but it might not be a deal on the other hand substituting threonine which is a non-polar amino acid for lysine would be a big deal this is non-polar this is basic that's a very significant change in chemistry and that will impact the function of the protein what are frame shift mutations to review this concept I've put together a sentence composed of three-letter words there are no spaces but we have these little dividers here if we were to do a mutation where we did a substitution of e for a over here then note that most of the words still make sense we have the cat what's a cat well you could probably surmise that that was intended to be cat and that it's just a typo but if we deleted one of the letters like hitting the delete key on your keyboard then what we get is if we drop this a over here then essentially we've significantly changed the meaning we have one word that makes sense and so far as it's a word but it doesn't make sense in the context over here that is called a frame shift mutation because codons are read in groups of three if you delete or insert you change the reading frame now let's look at some nucleotides here is a series of codons that code for four amino acids and then a stop codon and note that this example shows RNA to show the conso quences the mutation would have been in the DNA if we have a frame shift mutation then what we've done is we've done a deletion or an insertion that changes the reading frame just like we did over here and that will cause extensive missense or nonsense deleting this U over here causes these two amino acids to be wrong that's missense or what can happen is you can and have the insertion of a stop cat on and then the entire protein doesn't get Co for after this first amino acid that is the impact of a frame shift mutation CLE cell disease is caused by a single substitution mutation explain how one such substitution can cause CLE cell disease CLE cell disease is one of the first genetic diseases that was understood well on a molecular level it's important to know about the disease involves changes in the protein hemoglobin hemoglobin shown over here is a quary protein that carries oxygen in red blood cells so here's hemoglobin in red blood cells and here the red blood cells are carrying oxygen delivering it to the tissues of the body the mutation that causes CLE cell disease is a missense mutation and the eighth DNA triplet mutates from G A to CTC and that causes veine which is nonpolar to substitute for glutamic acid that causes a significant change in the chemistry of hemoglobin it causes hemoglobin molecules to stick together so notice how over here they're separate within the red blood cell but the mutated form they'll form very weak bonds and that happens under low oxygen conditions that just means when you exercise or walk up a flight of stairs anything like that that causes the cells to become sickled or spiked and they get stuck in red blood cells and that causes extensive tissue damage this is a recessive mutation you need to be homozygous in order to express the phenotype that there is a phenotype that is caused by being a heterozygote that's called CLE cell trait in any case this is how a single substitution mutation can be responsible for a significant genetic disease mutations can be positive negative or neutral explain the big idea is that a mutation effect always depends on the environment it's contextual a positive mutation improves a phenotype in a way that increases evolutionary Fitness Fitness is about survival and reproduction if it increases both of those things then it's a positive mutation here's an example this is a kind of fish that's called a thre spine stickleback there are populations that live in oceans there are populations that live in fresh water note this pelvic spine over here it's an adaptation that promotes survival in Marine environments because it protects against certain kinds of predators there are populations of sticklebacks that became stranded in freshwater lakes in those populations the Predators were absent there was a mutation that emerged that resulted in the loss of that pelvic spine not only because it doesn't make sense to produce a structure that has no survival benefit but there are predators that actually can prey on sticklebacks with the pelvic spine so losing the pelvic spine positive mutation we just talked about sickle cell anemia that's uh most cases a mutation that reduces Fitness why because it causes diseased red blood cells and it causes tissue damage as we just explained however in the context in which the mutation for Cle cell anemia evolve in some ways it's a positive mutation why because having one dose of the CLE cell alal in other words being a heterozygote gives you resistance to malaria so that makes it a positive mutation in a malaria ridden area in that context it's positive a neutral mutation has no effect on the phenotype and that's because it might happen in non-coding or non-regulatory DNA or it might result in a silent mutation where the amino acid doesn't change how are mutations important to evolution mutations provide the raw material upon which natural selection acts note that I'm using the same illustration as in the previous slide and I'm doing that on purpose mutation makes evolution a creative process that results in adaptation without mutation natural selection could only C harmful variants from a population but with mutation new variants can arrive rise that increase a population's fitness what's the difference between germline and somatic mutations germine mutations are mutations in the cells that make gametes and all other cells here's sperm here's an egg if there were a mutation in either one then that mutation would be present in every cell in the embryo that means it would be present in every cell in the organism and it would be present in the gametes that that organism produced germine mutations can be inherited they're subject to Natural Selection and what's an example any inherited genetic disease such as CLE Cel anemia which we have discussed or every adaptation a somatic mutation it emerges in some tissue during the course of development or during the course of adult life it only affects the organism it's not passed on on to the Future and an example are the somatic mutations that can cause cancer topic 6.7 part two horizontal Gene transfer here are some of the questions we'll be addressing contrast horizontal Gene transfer with vertical Gene transfer describe bacterial transformation bacterial conjugation and viral transduction what is viral Recon combination contrast horizontal Gene transfer with vertical Gene transfer in vertical Gene transfer parents transmit all or half of their genome to their offspring that's what's happening here here's a bacterium it's reproducing and in this generation all of the genes have been transmitted and in the Next Generation all of the genes have been transmitted you inherited your genes from your parents through vertical Gene transfer horizontal Gene transfer is quite different in horizontal Gene transfer one organism transfers genes to another organism that is not its Offspring that's what we see happening down here this bacterium over here is transferring genes on this Loop of DNA to this second bacterium in unicellular recipients as is shown here the newly acquired genes become part of the recipient's genome and and when this bacterium reproduces it'll pass on the newly acquired genes to its Offspring in a multi cellular recipient there's only longlasting results intergenerational results if the genes are transferred into the germ line describe bacterial conjugation if you want to build your vocabulary or use your vocabulary to enhance your understanding of biology conjugation is another word for sex here's how it works it's unlike any kind of sex that human beings or other animals have bacteria have in addition to their main chromosome main chromosome over here they have a loop of DNA that's called a plasmid plasmids can express genes for a membrane extension that's called a pyus that's shown over here at C when the pilis contacts a second cell the plasmid can be copied and transmitted to that recipient and the recipient now has all of the genes that are on the plasmid we have horizontal Gene transfer conjugation plays a key role in the spread of antibiotic resistant genes through bacterial populations describe bacterial transformation in bacterial transformation bacteria pick up DNA fragments shown over here at one from the environment and those DNA fragments enter into the cell and then become Incorporated into the genome that's what's shown over here this DNA can include plasmids so here's a circularized piece of DNA a plasmid that's being incorporated into the cell in genetic engineering transformation using plasmids is used to introduce foreign genes including human genes into bacterial cells describe how horizontal Gene transfer can occur through viral transduction transduction is a a kind of horizontal Gene transfer that occurs through viruses it occurs through mistakes in the viral replication cycle during viral infections the virus breaks apart the host's genome here's a virus it's injecting its DNA into its victim this cell over here and one of the first things that happens is that the cell that was infected has its DNA broken apart what the virus then does is it uses the cells mole ular Machinery to create new viral genes and to produce new viral proteins and those become assembled into new viral particles but during a mistake in the process sometimes DNA fragments from the host over here are mistakenly incorporated into a virus so this virus over here is leaving the cell carrying genes from the host it should just be carrying its own genes when that virus infects a cell in another or organism another bacterial cell in this case it can bring in the other organism's DNA so here's the DNA from the original host and notice that DNA is being injected into this new host that DNA will recombine with the DNA that's in the new host and this can happen to animals as well and if the virus infects a germline cell then new genes can be incorporated into the gene pool of the recipient what is viral recombination this is a kind of horizontal Gene transfer that occurs within viruses in this case two different viruses different strains of different viruses infect the same host so this virus and this virus they're variants of one another and here you see them infecting a new host and here you see both of these viruses inside a cell from the host as they carry out their replication cycle there's DNA from the host there's DNA from the viruses and the DNA from the viruses can get mixed up so the genes of the viruses can recombine and the result is instant emergence of new viral strains sometimes if animal immune systems can't recognize the new strain this can lead to pandemic viral out breaks this is what happens when every couple of years there's a strain of the flu that's new and that's novel and which infects many people sometimes with disastrous results this kind of viral recombination is what causes it is AP Bio making you feel overwhelmed and inadequate that's completely reasonable at learn biology.com we understand why students struggle with AP Bio the material is complex the pace is brutal and the vocabul is ridiculous but at learn biology.com we've created a way that makes it easier for you to study go to learn biology.com sign up for a free trial and complete our interactive tutorials and interactive AP Bio exam reviews we guarantee you a four or a five on the AP Bio exam see you on learn biology.com topic 6.8 biotechnology here are some of the questions that we'll be addressing what is Rec competent DNA how can it be artificially created how can you create recombinant bacterial plasmas that can express human genes what is gel electroforesis what is PCR what is sequencing explain what recombinant DNA is and how it can be artificially created recombinant DNA is DNA that's been combined from more than one source during meiosis you would create recombinant DNA as you combin the DNA that you inherited from your parents in the form of new gametes but this is artificial recombinant DNA it's DNA from more than one source shown over here that might have bacterial DNA with a snippet of human DNA with more bacterial DNA so DNA artificially combined more than one source it's been recombined the main tool in creating recombinant DNA is something called a restriction enzyme shown over here at letter C what these restriction enzymes do is they find sequences of DNA that're called restriction sites so here's one at B and they cut the DNA here's the DNA with sticky ends and you can see these sticky ends over here those sticky ends are exposed single strands of nucleotides you can see that over here in this diagram that shows the DNA double helix and those single strands are able to form hydrogen bonds with complimentary bases the result of using restriction enzyme shown over here shown over here are restriction fragments so here's a fragment over here here's another fragment over here if you cut a second piece of DNA for example a piece of human DNA with the same restriction enzymes what you'll wind up having are complimentary sticky ends because of complimentarity the ends of the two pieces will form hydrogen bonds that's what you see happening over here and then you need to use another enzyme it's called DNA ligase and that creates sugar phosphate bonds connecting the strands creating recombinant DNA using restriction enzymes and DNA ligase you can create a recombinant plasmid with a human gene explain how note for this question question assume that introns have already been removed from the human DNA the first step is to extract a plasmid from a bacterial cell and then cut open that plasmid with restriction enzyme leaving sticky ends the way that we just described in the previous slide use the same restriction enzyme to cut out a Target human gene and therefore the ends will be complimentary because they've been cut with the same restriction enzymes the human gene will combined with the plasmid forming hydrogen bonds between their complimentary sticky ends then you have to use DNA liase we referred to that in the previous slide it's not shown here to bind the human DNA and plasma DNA together creating a recombinant plasmid that contains a human gene then you'd insert the plasmid into a bacterial cell that's using the technique of transformation which we previously referred to when we talked about horizontal Gene transfer this genetically engineered recombinant bacteria this over here and its descendants over here will produce the human protein and produce the plasmid in every reproduction cycle this is how genetically engineered insulin has been created that means bacterial cells that produce a human Protein that's widely used in order for the genes for human protein such as insulin to be expressed in bacteria introns need to be removed explain why and how to review introns are non-coding sequences of DNA within eukariotic genes that have to be spliced out before the genes RNA can be translated into protein here's human DNA there are exons that are expressed sequences and they're separated from one another by introns these intervening sequences the consequence of the presence of ventron is that to transfer a human gene to a bacterium to create a gene product you have to use DNA from which the introns have been removed the bacteria would just translate everything including the introns and that would lead to a nonfunctional protein how do you remove the introns you have to do it before transforming the bacterial cells and you can do it in two ways the first method involves determining the amino acid sequence for the protein biochemists can look at a protein like insulin and figure out what the linear sequence of amino acids is once you do that you use your genetic code chart to reverse engineered DNA that codes for that amino acid sequence another method is shown here what you do is you find cells that produce the desired protein you extract mRNA from those cells that codes for this protein that that mRNA already has had its introns removed and then you use the enzyme reverse transcriptase that's an enzyme that's in retroviruses which are viruses that are RNA based but can create a DNA copy of themselves that gets incorporated into the human cell that they've affected HIV is an example of one such virus you use reverse transcriptase which is shown here at B to create cdna compliment DNA from the RNA and then you insert that complimentary DNA into the plasmid and that's how you do your successful genetic engineering what is gel electroforesis how is it used to analyze DNA gel electropheresis is a technique that's widely used it's used to sort molecules by size and or electrical charge it's the basis of a technique that's called restriction fragment analysis also called DNA fingerprinting widely used in forensics it involves placing molecules in a porous gel here's the gel over here at number five that is in a device an apparatus a box that can produce an electrical current so you'd run an electrical current generated over here through the gel because DNA's phosphate groups shown over here are negatively charged DNA fragments will move away from the negatively charged side of the electroforesis chamber so you put the DNA over here like repels like negative charge negative charge and that's going to push the DNA in this direction the small fragments will be impeded by the gel less than the large fragments so over time the smaller fragments will move more than the larger fragments enabling the fragments to be sorted by size by the end of the process you'd have here DNA with one large fragment here DNA with two fragments and here DNA that's been cut into three fragments how would it be cut by restriction enzymes so you use a combination of these techniques to get results like this as you're analyzing DNA material related to biotechnology often shows up on the AP Bio exam in this form here's a simple restriction mapping problem a 20 kilobase plasmid KB kilobase has several restriction sites the image on the right shows the results of electroforesis following various combinations of restriction enzymes which lane shows the gel that would result if the plasmid were digested with the Restriction enzyme bam H1 this line over here indicates a restriction site that's been l labeled with bam H1 so there's a restriction site over here for bam H1 a second one over here and a third the entire plasmid is 20 kilobases and the map is telling you that this is a 3 kilobase difference so if you cut the plasmid with bam H1 you'd wind up with three fragments here's one here's a second one would start here go all the way down to here and here's a third one that would start here here go all the way up to here the first one would be 3 kilobases in size the second one would be 11 kilobases in size how did I do that 3 kilobases + 8 = 11 and the last one is 6 kilobases in size and what that means is that you'd have to look over here at the gel and you'd see oh this fragment is 11 kilobases this fragment is six kilobases and this one is three per perfect that means B would be your answer if this were on a multiple choice test what is PCR what is it used for PCR stands for polymerase Chain Reaction the polymerase is DNA polymerase it's a cellfree technique for cloning DNA in other words you can clone DNA in a test tube you don't need a cell in order to do it it requires the DNA sample that you want to clone it's shown over here at a it requ re Ires primers those are short strands of single stranded DNA that bind to sequences at the start of the DNA that you want to amplify so here is a primer and here you see the primer binding to the Target DNA it requires heat resistant DNA polymerase shown over here at G that would be a large protein why does it need to be heat resistant because the process involves repeated cycles of heating and Cooling and you need a DNA polymerase that won't be denatured by the heating process where do you find it from bacteria and archa that live in hot springs and you also need free nucleotides that are going to be used for DNA synthesis because what we're doing is we're making lots of DNA from a sample that we want to amplify how does it work it involves repeated cycles of heating the DNA to separate it into different strands so here's the DNA you heat it youing break those hydrogen bonds and now you've separated it into single strands that's step one over here then you cool the DNA enough so that primers can bind to it and so that DNA polymerase can synthesize new DNA that's shown at two and it's shown at three the DNA polymerase will read the template Strand and it'll seal uh sugar phosphate bonds between the nucleotides that bind with the template strand so here you are uh you're seeing DNA poates creating new DNA and every Heating and Cooling cycle will double the amount of DNA so we started with one piece of DNA now we have two pieces of DNA that's an exact copy of the original DNA do it again you have four pieces of DNA do it again and you'll have eight pieces of DNA after 10 Cycles you have a thousand times more DNA than you started with and after 30 Cycles you've Amplified your DNA ail millionfold this is widely used in any kind of science that needs to work with DNA it's widely used in forensics where little DNA samples from a crime scene for example are Amplified so that they can be analyzed for electroforesis DNA fingerprinting Etc what is DNA sequencing what are some of its uses DNA sequencing you just need to know what it is you don't really need to know how it's done though if you're interested in seeing how it's done you can do that at learn biology.com DNA sequencing involves taking a sample of DNA anything from a small fragment to the entire Genome of an organism and figuring out the specific sequence of a t c n g nucleotides that make it up it allows biologists to determine what proteins an organism can produce it's used to infer evolutionary relationships and it's used by cancer biologists to sequence tumors to see what genetic mutation are causing the cells to become cancerous during the covid-19 pandemic sequencing was used to analyze the emergence of new SARS K2 variants and of course it was used in order to create the vaccine in forensic sequencing is being used along with DNA fingerprinting to identify and exonerate suspects and resolve paternity disputes want to learn more sign up for a free trial of the website that guarantees your AP biology success learn biology.com and watch this next video