[Music] [Music] normal Gene function is is to code for a particular protein uh here we're going to be looking at the genetics of microbes looking at mainly the circular DNA of bacteria and looking at differences between procaryotic and eukaryotic DNA again remember that ukar outs and procaryotes possess chromosomal DNA eukariotic DNA is linear and exists inside of the nucleus a double membrane called the nuclear envelope inside UK carots normally have several chromosomes linear chromosomes conversely bacteria typically have a single circularized chromosome that exists as a structure called the nucleoid and that nucleoid is floating around in the cytoplasm notice how there is no nuclear envelope surrounding this DNA the DNA is floating around directly in the cytoplasm again UK carots have multiple linear chromosomes here's a chromosome here's a chromosome and bacteria have a single circularized chromosome and of course our chromosomes whether it be UK carots or procaryotes consists of double stranded DNA when we refer to the genome we're referring to all of the genetic material of an organism most of our genome exists in the form of chromosomes for instance humans typically have 46 chromosomes however remember we also have what's known as extra chromosomal DNA this is DNA that lies outside of the nucleus this DNA Falls inside various organel like our mitochondria do you remember when we discussed the mitochondria we said that the mitochondria possess their own single circularized chromos plants also contain chloroplasts in addition to mitochondria and that chloroplast also contains its own single circularized chromosome so these would be examples of extra chromosomal DNA DNA that's not part of our nucleus not part of our chromosomes but is technically part of our genomes in addition several UK carots but mainly Pro carots possess what are known as plasmids tiny pieces of circularized DNA these plasmids we're going to go into these plasmids in more detail but they can have important genes uh on them they can have important genes with important functions for the organism and these plasmids can be passed from organism to organism giving different organisms the traits we're going to talk about what plasmids are but remember plasmids are tiny pieces of extra chromosomal DNA that are typically found in procaryotes but can be found in ukar Nots as well now the genomes of all cells consists of DNA all living organisms have genomes of DNA however viruses which are not alive can contain genomes of DNA or RNA but not both and the study of genomics is the study of an organism's entire genome again if we're looking at a eukaryotic cell we have the nucleus which is a double membrane comprising the nuclear envelope and inside we have linear chromosomes humans would have 46 of these linear chromosomes typically outside of the nucleus we're talking about non-chromosomal DNA like the DNA found in our mitochondria which is a single circularized chromosome the DNA of plants and algae uh which is again a single circularized chromosome and various plasmids which are tiny circularized pieces of DNA as well in bacteria and in Pro carots in general you typically have a single circularized chromosome which is called the nucleoid and outside of that you have this non-chromosomal DNA called plasmid DNA small circularized dnas with a handful of genes on them and remember that viruses which are not alive can have their own genomes consisting of either DNA or RNA but not both recall that a chromosome is defined as a distinct cellular structure composed of a neatly packaged DNA molecule so think of it as a single doubl stranded DNA molecule that's part of an organism's genome inside of the nucleus if it's a UK carot or as part of the nucleoid if it's a procariota chromosomes are located in the nucleus of the cell they vary in number from a few to hundreds remember humans have 46 chromosomes but different organisms have different numbers of chromosomes and eukaryotic chromosomes are linear in appearance that means each chromosome has a beginning and an end remember again bacterial chromosomes usually bacteria have a single circularized double strand DNA chromosome but that's not always the case but for the purposes of this lecture you know we're going to be discussing bacterial chromosomes as a single circularized chromosome and remember the difference between genes and chromosomes genes are basic informational packets in which a chromosome is subdivided containing the necessary code for a particular cell function in general a gene is a segment of a chromosome that typically codes for a protein for instance humans may have 46 chromosomes but those 46 chromosomes house 25,000 genes you can think of a gene as just a segment of a chromosome that usually codes for proteins that's not the entire picture but we could simplify it to that definition for now also recall the terms genotype and phenotype genotype refers to the genes or various alals that an organism possesses alos are simply versions of a gene so for instance eye color would be a Jee blue or brown eyes would be Al of that Gene you can think of alals as versions of a gene and the alals that you possess that you inherited from your parents make up your genotype so for instance my genotype might be brown eye color brown eye color uh and I inherited one of those Al from each parent so my genotype would be brown brown however your genotype might be brown blue you may have inherited a brown eye color alil from your mom and a blue eye color alil from your dad so your genotype might be different from my genotype however the phenotype is the expression of the genotype it's the trait that we see for instance if you see that I have brown eyes that's my phenotype notice how phenotype is slightly different than genotype my phenotype is brown eyes because I have brown eyes however my genotype could be brown brown because I inherited a brown alil from both parents or brown blue because I inherited a brown eye color alil from one parent and a blue eye color alil from the other parent however procaryotes like bacteria they don't have parents cuz there is no sexual reproduction that occurs in bacteria or procaryotes for that matter like ARA and so a procaryote or one Alo for each phenotype you don't have parents so they don't have two sets of chromosomes they don't have two Al for every Gene now this leads to variation in genome size eoli for instance eishia coli remember it has a single circularized chromosome well that single chromosome houses approximately 4,000 genes humans on the other hand remember we have close to 25,000 genes residing on our 46 chromosomes so different organisms have different numbers of genes and genes typically code for proteins here you can see an actual image of DNA under a transmission electron microscope remember that DNA is made up of build blocks these are monomers called nucleotides DNA and RNA are made up of building blocks called nucleotides every nucleotide has three main components a phosphate group a deoxy ribos sugar and a nitrogenous base the only difference with RNA is that there is ribos sugar instead of Di oxy ribos sugar and in RNA instead of the nitrogenous based thyine we have the nitrogenous base uracil otherwise the basic unit of DNA is the same for DNA and RNA again in DNA we have phosphate group deoxy ribos sugar nitrogenous base these three components make up the nucleo of a DNA molecule and if you've ever heard of the genetic code you know how you may have heard that the genetic code is made up of A's G's T's and C's well that has to do with the nitrogenous bases of the DNA molecule the nitrogenous base can exist as adenine or a for short thyine or t for short guanine or G for short or cytosine C for short these are the letters that make up the genetic code and they are different versions of nitrogenous bases on these nucleotides and when you string nucleotides together this is how you obtain a strand of DNA and you may remember from previous courses that each strand of DNA has has a five Prime end and a thre Prime end and what's important to remember is that the two strands of DNA that make up the double helix of DNA those strands are anti-parallel this means that for instance this blue strand of DNA has its five Prime end here and it's three prime end at the other end but the the complementary red strand would be going anti-parallel would be going in the opposite direction with its five Prime end down here and its thre Prime end up here so we say that double stranded DNA consists of two complimentary strands of DNA and those two complimentary strands are antiparallel with one another remember that each strand of DNA has a sugar phosphate backbone you see here in highlighted in this peach color there is the deoxy ribos sugar followed by a phosphate group sugar phosphate sugar phosphate sugar phosphate this is known as the sugar phosphate backbone of DNA and notice poking out the sides are the various nitrogenous bases T's G's C's and A's and on the complementary strand of DNA which is anti-parallel notice it also has a sugar phosphate sugar phosphate backbone and its own nitrogenous bases a c g and T the two strands are held together by what's called complementary base pairing A's pair with T's with two hydrogen bonds C's pair with G's with three hydrogen bonds and so so on and so forth remember when I said each strand of DNA here you have two strands of DNA each one has a five Prime end and a thre Prime end we'll recall that the five Prime end of each strand of DNA has a phosphate group hanging off while the three prime end of each strand of DNA has a hydroxy group hanging off hydroxy is an O attached to the three prime carbon of this deoxy ribos sugar and there you have it the double strand DNA I know that some of this may seem confusing especially if you haven't had biology 1406 or a bio1 course recently because a lot of this that I just mentioned uh assumes that you've had some background in this so if you haven't just please make sure to read that part of the book so that you could become up to speed now I told you that DNA or genes code for proteins and that's typically what a gene does it codes for a particular protein so the process by which information is transmitted is that genes code for the proteins those genes are copied into what is known as mRNA or messenger RNA which is essentially an RNA copy of that Gene and then ribosomes can interpret that mRNA message to produce a protein this flow of information from Gene to RNA to protein is called the central dogma of genetics the first step to the central dogma is called transcription this is where the DNA or the gene is copied to mRNA to synthesize a new strain of mRNA remember that mRNA stands for messenger RNA and that messenger RNA is a single stranded RNA copy of the gene next during translation that mRNA message is read by your ribosomes to produce a protein that's what a ribosome does you can think of a ribosome as a 3D printer that prints proteins based on the message found on the MRNA and by the way the process of making proteins from genes is called gene expression G the first step to gene expression is transcription where a RNA polymerase copies the gene from DNA to a copy mRNA messenger RNA and the second part of gene expression is called translation where ribosomes Chomp down and they read the message of the MRNA from five Prime to three prime synthesizing a protein based on that message so again what is the central dogma of genetics the central dogma of genetics states that information flows from Gene to mRNA to protein here you can see gene expression at work we're starting with the circularized chromosome of a procaryote a gene is then transcribed a gene is copied into mRNA let's say this this examp example here in the center messenger RNA which is then translated by the ribosomes into a protein remember that RNA polymerases are responsible for transcription while ribosomes are responsible for translation so again we say that genes give us our traits but that's only part of the story it's actually the proteins which give us our traits the genes merely code for those proteins so for instance if I have brown eyes it's not because the gene is directly causing my eyes to be brown what's happening is the brown eye color Alo is being transcribed by RNA polymerase into messenger RNA and then ribosomes read that messenger RNA to produce a protein that protein produces pigment in my eye and makes my eye appear Brown so it's the protein that results from my brown eye color Al or Gene that's actually making me have that trait making my eyes brown so you can think of it as proteins are what really give you your traits but the only reason your cells know how to make those prot proteins is by having the genes and expressing those genes into proteins so if that's true then proteins ultimately determine your phenotype how you look what traits you have and there's a whole new field of biology known as proteomics which is the study of an organism's complete set of expressed proteins we want to know what all of those proteins do in your body and again it's your DNA that is the blueprint that tells the cell how to make those important proteins again to simplify and overview gene expression remember that genes consist of double stranded DNA this is simply a segment of your chromosome that Gene is made up again of double stranded DNA but the template strand the template strand is the Strand that is copied by RNA polymerase into a single strand of Mna messenger RNA shown here in green and it's then those ribosomes which can read through this message this mRNA message to produce the proteins and do you recall that proteins are made up of building blocks called amino acids there are 20 important amino acids that make up the proteins of your body those those amino acids are strung together by the ribosome to form a chain of amino acids and that is what a protein consists of now I want you to note here that every three nucleotides on the MRNA transcript see nucleotide 1 2 and 3 1 2 3 1 2 3 every three nucleotides on the MRNA transcript code for a particular amino acid and these blocks of three nucleotides has a name they're called codons and various codons are deciphered by your ribosomes to construct your proteins in fact let me show you this this is known as the codon table and notice that there are 64 three-letter words in the genetic language 64 codons if you will so for instance CCU the code on CCU is translated by your ribosomes and from mRNA to mean the amino acid Proline whereas UCA is deciphered by your ribosomes as the acid Serene notice how you have these threel words these codons and each codon has a meaning most codons code for amino acids but there are a handful of very important codons let's start with this one here this codon which reads A U is a special codon the first a ug that the ribosome encounters is called the start codon it instructs the ribosome to start making a protein and not only does it do that but it tells the ribosome that the first amino acid should be the amino acid methionine so yes every protein should start with methionine because the start code on itself instructs the ribosome to put down the amino acid methionine the ribosome will then read every codon between the start codeon until it reaches a stop codeon the stop codons there are three different ones but any one of the three is sufficient to stop translation the stop codons include UAA u a or UA any one of the three will tell the ribosome to stop making a protein so isn't that neat the entire genetic code can exist on a postage card it's 64 codons and those codons make up the genetic code they make up the genetic language they're responsible for all the proteins which make up all the diversity of all of life isn't that neat to think about and the ribosome is the translator of the code it knows how to read the genetic code and to build the correct proteins which are Pres described now for the first major difference I want you to note between gene expression in UK carots versus procaryotes take a look here remember this is a eukaryotic cell and remember that the chromosomes are within the nucleus this means that transcription occurs in the nucleus first that's where the genes are transcribed to mRNA and then if you remember from an early biology class biology 1406 for us at that point that mRNA needs to be processed do you remember the three steps to mRNA processing splicing out of inance adding a five Prime cap adding a three prime poly a tail this is known as RNA processing and it has to occur after the MRNA is formed then that processed mature mRNA is then allowed to leave the nucleus meet up with the ribosomes where translation can finally occur in the cytoplasm did you follow that transcription and RNA processing occur first in the nucleus and then transl occurs later on in the cytoplasm now I want to show you something in a bacterial cell the nucleoid the genes are right there in the cytoplasm so transcription and translation happen in the same place they happen in the cytoplasm there's no separation of the DNA from the ribosomes they're both in the cytoplasm and and not only does transcription and translation happen in the same place the cytoplasm but it happens at the same time because RNA processing is not necessary in bacteria so let me show you an image uh depicting what I'm talking about on this slide we have the protein assembly line of bacteria this is what I'm talking about take a look here remember that the DNA is floating around as a nucleoid in the cytoplasm the RNA polymerase enzyme copies a particular gene into a transcript in Orange you see this is the MRNA transcript it's called and look what's happening the ribosomes which remember ribosomes consist of a large subunit as well as a small sub subunit the ribosomes Chomp down on the five Prime end of the MRNA and then they proceed to search for that start codon so that they can start making a protein notice how transcription and translation are occurring at the same time in the same place notice how transcriptions not even done yet RNA polymerase is still doing its job RNA polymerase is still constructing a strand of mRNA before RNA polymerase is even done making the MRNA the ribosomes are free to attach and search for the start codon to begin translation isn't that neat so that this should also tell you something else not only can transcription and translation occur at the same time in the same place but RNA processing is not necessary in bacteria this means there's no removal or splicing out of the introns there is no five Prime cap there is no three prime poly a tail the transcription and translation occur at the same time same place and it's very efficient isn't it in fact it's so efficient that that when the when the ribosome attaches and starts reading the MRNA the next ribosome can attach and then the next ribosome attaches and then it pretty much becomes a Congo line of ribosomes and so like I said before before the the RNA polymerase has even completed transcription a Congo line of several ribosomes this is called a poly ribosomal complex can attach to the MRNA and they all search for that start codon and translate that message to protein here in purple this is the protein in purple until they reach the stop codon any one of the three stop codons in which case they have produced protein so protein synthesis in bacteria is very efficient it transcription and translation occur at the same time in the same place and RNA processing is not required because for one reason bacteria do not have what are called introns they don't have this intron junk DNA they used to call it or fer DNA inside of the genes to splice out isn't that neat so gene expression in bacteria is very very quick and very very efficient now for our second big difference between gene expression in bacteria versus gene expression in ukar Nots and this is the presence of what are known as operons operons are coordinated sets of genes regulated as a a single unit operons are only found in bacteria and archa so they are specific to procaryotic cells and how does it work what exactly does it mean to have a coordinated set of genes let me explain what an operon is at the board and we'll come back to go into some details about different types of common operon found in bacteria now before I explain what an operon is let me show you how genes work in you and me in UK carots when it comes to a UK carot we have a gene and remember a gene is a unit of heredity that codes typically for a protein and in order to express this Gene remember there is a enzyme called RNA polymerase RNA polymerase attaches in front of the gene it's called Upstream of the gene at a site called the promoter yes Gizmo Gizmo are you making an appearance on a live shot here come here Gizmo yeah Gizmo you guys are finally seeing Gizmo In the Flesh Gizmo wants out he wants to go find Wicket and play anyway just uh we did not expect this little break time with Gizmo but this is my buddy Gizmo and he wants out so say bye Gizmo come on guys all right go play go find wicket so sorry again in you and me we have genes genes code for proteins the way they do is first by a process known as transcription where the RNA polymerase enzyme binds to the promoter and then proceeds down the gene transcribing the gene as an mRNA the MRNA is a single strand of RNA that is a complimentary copy of the template strand of the Gene and then remember the next step we have translation where the ribosomes will construct protein from that mRNA message okay and remember this is no known as gene expression the first step being transcription and the Second Step being translation and remember what I mentioned this Gene codes for a particular protein and that Gene is transcribed to mRNA which is translated to protein and that makes sense you and I are UK carots each Gene is transcribed as an mRNA which is translated to protein but now let me tell you the difference in procaryotes and the fact that procaryotes have a variation on this concept known as operons and what is an operon so let me explain that next now operon are only found in procaryotes and you can find them in bacteria and what an operon is is a series of functionally related genes one right after another so on your chromosome remember bacteria have a single large circular chromosome they might have three genes in a row for instance Gene one gene two and Gene three these might be three different genes but they are they code for functionally related proteins what does that mean that means the the protein product from this Gene and this Gene and this Gene might actually all synergize and do the same function um they might all produce help produce the same component for the cell or they might all help break down the same nutrient in the cell okay so for instance the most famous and well studied operon is known as the Lac operon or lactose operon and it's three genes that all serve to transport and break down lactose and what's cool is these three genes for instance might have a singles shared promoter in front so you see instead of each gene having its own promoter like you and me do it may have a single promoter Upstream of three genes this means that when RNA polymerase when RNA polymerase binds to the DNA and proceeds to transcribe and proceeds to transcribe it actually transcribes Gene one then it transcribes Gene 2 and then it transcribes Gene 3 and then it stops this means that you end up with a super long mRNA this mRNA is very long because the MRNA is even though it's one long mRNA it has information for three different genes which means it possesses the information to synthesize three different proteins through translation so during translation during translation a ribosome might attach here a ribosome can attach here and a ribosome can attach here and this ribosome will translate this protein this ribos Rome will translate a different protein and the third ribosome will translate a third protein so effectively we have synthesized three different proteins at the same time isn't that neat and remember these three different proteins may all have the same job like transporting lactose and then breaking down the lactose you see so they all function in a in a similar way uh they all serve to do a similar function so there are different operon that we're going to learn about like the lactose operon or the Lac operon for short there's the Arginine operon or the arge operon for short uh there are many different operon and now you know how they work now again I want you to know that we do not find operon in UK carots so you and I we don't have such setups we don't have three different gen that could all be transcribed as one long mRNA right that's what an operon is and it's only found in proc carots welcome back so now you know what operon are operon are sets or coordinated sets of genes all regulated together as a single unit this means that a really long mRNA is constructed and that mRNA contains more than one Gene and those genes are functionally related so let's talk about how these operon are controlled in bacteria operons can either be what are called inducible or turned on or repressible turned off so let's talk about inducible operon and why they're important followed by repressible operon opon and and how they could be important for the cell as well inducible operon contain genes that make proteins that help metabolize nutrients so what is an inducible operon an inducible operon is an operon that is normally not expressed it is not transcribed and translated normally however the operon can be turned on that's what induced means so again the operon is normally off but it can be turned on or induced and it is usually induced by a nutrient for which those genes encode and it is normally turned on by the nutrient which those genes break down so I'll give you an example the most famous and well-studied example of an inducible operon is called the lactose operon or Lac operon for short if there's no lactose around the bacteria has no reason to make the enzymes to break down lactose however what if lactose is around we want to break down that lactose for sugar right well if lactose is around the lactose can serve as an inducer turning on the genes that break down lactose isn't that interesting again imagine if you're ecoli and there's no lactose present you don't want to spend the resources to express the lactose operon and spend all those resources to transcribe and translate the three Lac operon genes to make the proteins that help break down lactose you don't want to make those proteins if there's no lact toose around it's expensive to make proteins it costs resources amino acids ATP it costs the cell resources to make enzymes why make the enzyme that breaks down lactose if there's no lactose in the environment you see what I'm saying so typically inducible operon contain genes that make proteins that help metabolize nutrients nutrients like lactose these operon are normally off so if there is no nutrient around then the operon is off but if the nutrient is around like lactose is around then that turns those genes on so we can digest that nutrient isn't that interesting so again enzymes needed to metabolize a nutrient such as lactose are only produced when that nutrient is present in the environment so let me actually show you how this inducible operon works let me show you how the lack operon works and then we'll talk about the repressible operon the Lac operon consists of three functionally related genes in this case Gene one gene 2 and Gene three all three of these genes help to metabolize lactose in fact one of these genes is the gene called beta galactosidase which results in production of the protein Beta galactosidase And this is the protein enzyme that breaks down lactose so the Lac operon consists of three genes all that help to break down lactose the Lac operon has what's called the promoter remember the pr motor is this stretch of DNA Upstream of the genes on which RNA polymerase the enzyme that reads DNA to synthesize mRNA can bind RNA polymerase binds to the promoter so that it can start transcription however there is a stretch of DNA between the promoter and the genes and this stretch of DNA is called an operator the operator can accommodate this guy look at this thing right here this is known as a repressor protein it's drawn kind of like a clothes pin in this image and I like that because imagine imagine this repressor protein is a closed pin if I were to attach a repressor protein to the operator and you know just like this Clos pin looks here on this line I want to ask you this if the RNA polymerase attaches to the promoter and then it proceeds to go this way wouldn't it run into that repressor and wouldn't that repressor get in its way and prevent it from traveling down the three structural genes and transcribing those as an mRNA unit and that's exactly right look the repressor protein binds to the operator preventing RNA polymerase from Pro seeding down the operon so this prevents transcription from occurring this means that the operon is effectively off and remember these inducible genes are normally off so how do we turn this operon on which means allow RNA polymerase to do transcription let's talk about that look at step two if lactose is added added to the cell's environment it triggers events that turn the operon on you see these two purple dots right here these two purple dots represent the disaccharide lactose and it turns out the that the lactose itself the nutrient itself serves as the inducer for this operon how can that be take a look the lactose disaccharide it directly binds to that repressor protein and when it does watch look what happens the the repressor becomes inactive which means upon lactose binding this changes the confirmation changes the shape if you will of the repressor allowing the repressor to leave the operator now let me ask you this if the repressor leaves because it binds lactose wouldn't that allow the RNA polymerase to continue down the structural genes and transcribe Gene one gene 2 and Gene 3 as one long mRNA molecule well it will so now you have effectively turned on the Lac operon by adding lactose the inducer the lactose inactivated the repressor allowing RNA polymerase to transcribe the three genes and remember because this is an operon all three of those genes are expressed as one single very long mRNA which is then recognized by ribosomes the ribosomes Chomp down in front of all three of the genes in this long mRNA to form how many different proteins can you beat Wicket how many different proteins are made from this single long mRNA how many different proteins can be made that's right Wicket as always very smart cat uh three different proteins remember three different genes are expressed or transcribed transcribed as one long mRNA ribosomes will construct three different proteins from that long mRNA and all three of these proteins including beta galactosidase are important to not only transport the lactose but to digest the lactose into monosaccharides glucose and galactose isn't that neat so now the the ecoli or the bacterium whichever bacterium it is is free to digest that nutrient and whenever lactose becomes deficient in the environment let's say the lactose levels drop off then the lactose will detach itself from the repressor the repressor will again activate bind down on the operator turning off gene expression of this operon isn't that neat so again the lactose operon is induced by lactose and it can be uh turned on with the addition of lactose remember inducible operon are normally off if that nutrient is not present but can be turned on in the presence of that nutrient and why is that so that we can conserve resources when we don't have that nutrient around now before we discuss repressible operon I realized it's time for a break time with our favorite cats Gizmo and Wicket let's see what these guys are up to and we'll be right back with repressible operon [Music] whereas inducible operon are normally off but can be turned on repressible operon are normally on but can be turned off that's what repressed means it means to turn off so their normal state is on and usually repressible operon contain genes coding for the enzymes that synthesiz a particular amino acid remember that there are 20 amino acids and all 20 are necessary for building proteins so bacterium they need to make these amino acids all the time however if a bacterium comes across a windfall of a particular amino acid then it may not need to make anymore at the time and it can shut off the production of the enzymes that produce that amino acid so for instance what if I have eoli in a broth and that ecoli is making a particular amino acid what if I provide that amino acid like I I inject that amino acid into its environment and now it has plenty of that amino acid well in this case the presence of that surplus of that amino acid can repress the operon for that enzyme to make that amino acid because why waste the resources to make the enzymes that synthesize that amino acid if there's plenty of that amino acid in the environment you see what I'm saying if there's not much of that amino acid in the environment you need to synthesize your own by expressing these enzymes however if you have plenty of that amino acid in the environment you could save Resources by repressing the operon for these enzymes here's an example of a repressible operon this is known as the ARG operon or Arginine operon so remember as with all operon you have numerous genes that are functionally related in this case this is the Arginine operon so these genes are expressed to form proteins that function to synthesize the amino acid arginine one of the 20 important amino acids for making proteins so the way this works is the operon is normally on which means RNA polymerase is free to bind to the promoter proceed down and transcribe structural genes 1 2 and 3 remember a very long mRNA molecule is transcribed which contains the information for all three genes ribosomes attach to translate all three genes and how many different proteins are made again can you beat Wicked that's that's right as always Wick it three different proteins are synthesized check it out protein 1 protein 2 and protein 3 are all created from that mRNA and remember the function of these proteins is to synthesize Arginine Arginine looks like these little stars and these Arginine amino acids are free to be used in metabolism they're free to be used to make more protein okay so notice how the operon is on and the reason the operon is on is because the Arginine repressor the AR the Arginine operon repressor is inactive it's inactive it does not bind to the operator normally so how does this operon turn off let's discuss that how is this operon repressed remember this operon can be repressed if I were to spike in a bunch of arginine imagine if the microb encounters a high concentration of arginine so let's discuss what happens if Arginine accumulates you have a high concentration of arginine at some point when you reach this high threshold concentration of arginine Arginine will bind to that inactive Arginine rep repressor protein and activate it now because of binding to Arginine the repressor is active in this case they call Arginine a co- repressor because it helped the repressor become activated so the co-repressor Arginine binds to the active repressor protein and converts it to the active form the active repressor serves as a clo pin remember kind of like a cloth pin on a dryer line it binds to the operator and look what it's done it's gotten in the way of RNA polymerase RNA polymerase might proceed but it will hit the operator it will hit the repressor which is on the operator and transcription will be off this means that the Arginine operon is now off transcription is blocked Arginine synthesis is arrested it makes sense if there's plenty of arginine we don't want to waste the resources to synthesize these Arginine making genes okay so again this is an example of a repressible operon so now let's discuss the concept of recombination in bacterial cells recombination is an event in which one bacterium donates DNA to another bacterium remember that bacterium they don't reproduce with sexual reproduction however they can exchange DNA and this is known as recombination and the end result of recombination is a strain of bacterium that's different from both the donor as well as the recipient often times the DNA that's shared is a plasmid remember a small extra chromosomal DNA that can move between cells but it doesn't have to be a plasmid bacteria can also share genomic DNA as well so the term recombinant refers to any organism that now contains and expresses genes that originated in another organism we discussed this briefly before remember that animals reproduce by sexual reproduction when the male and the female meet there is an exchange of genetic information and that Offspring has genes from both parents that passing on of genetic material from parents to offspring is called vertical Gene transfer but remember that bacteria can donate genetic information from one bacterium the donor to another bacterium the recip ient and remember this is not called vertical Gene transfer this is known as horizontal Gene transfer and we briefly mentioned this in a previous chapter remember when we talked about the sex pilus and how certain bacterium can form a sex pilus and then send some genetic information to the the recipient cell through that Hollow tube that's an example of horizontal Gene transfer any transfer of DNA that results in organisms acquiring new genes that did not come directly from parents so again bacterium they share genes with horizontal Gene transfer whereas animals we share our genes through vertical Gene transfer now the sex pilus and conjugation are not the only form of horizontal Gene transfer so we're going to talk about the the three different forms of horizontal Gene transfer cuz that's not the only one and again often times it's this small circular piece of DNA called the plasmid which is shared from one bacterium to another these plasmids they contain at most just a few dozen genes but those genes May confer some advantageous traits to that organism what kind of advantageous traits for instance the F plasmid or fertility plasmid as it's known is a plasmid that encodes the genes necessary for constructing your own sex uh pilus for constructing your own uh sex pilus for conjugation you can also have what are known as resistance plasmids or R plasmids these for antibiotic resistance to the bacterium that possess them there are also virulent plasmids for toxins and such where the plasmid may contain genes that encode toxins these plasmids often carry useful traits now remember I mentioned there are three types of horizontal Gene transfer in bacteria and we touched on conjugate ation however there are two more forms transformation and transduction as well all three are common forms of horizontal Gene transfer in bacteria so we're going to discuss each the first type of horizontal Gene transfer is called conjugation remember this is done through the sex pilus and through that pilus either a plasmid or other genetic material which can include a part a portion of the Genome of the bacterium can be sent from the donor to the recipient now it's important to understand that this is a oneway exchange of DNA not a two-way exchange so DNA is sent from the donor cell the cell that constructed the sex pilus and it and that DNA is s to the recipient cell the recipient cell does not send a genetic information back to the donor and conjugation can occur in both gram positive and gram negative bacterium and one of the best understood conjugative plasmids is called the F factor in eoli remember the F factor is also known as the fertility Factor it's a plasmid that contains the genes necessary to construct the sex pilus so here we can see conjugation using the F Factor take a look here there are two forms of conjugation with this F Factor there is what's called f Factor transfer and HFR transfer let's go through each for transfer of the F Factor here is the scenario here is the donor bacterium on the top and here's the recipient at the bottom the squiggly blue and purple lines here the these represent the Genome of the bacterium remember that the bacterium have a single circularized DNA in the form of nucleoid here is the nucleoid of the donor of the donor bacterium here is the nucleoid of the recipient and in addition you see this small circular DNA this represents the F factor or the fertility factor a small circular plasmid of DNA and what's interesting about this F plasmid again is that it contains the genes necessary to make this bridge to make the sex pyus so the donor cell is called the f plus cell because it contains the F plasmid this donor cell constructs a sex pilus which docks onto a neighboring cell and by the way this neighboring cell does not need to be the same species it could be a different species uh of of cell now once the bridge is made with the pyus the plasmid is copied and a copy of the plasmid is sent to the recipient cell this is known as rolling Circle replication this is how the plasmid is copied and a copy of that F factor is sent to the the recipient cell and remember in conjugation genetic information only goes from the donor cell to the recipient cell and in this case a copy of the F Factor plasmid is given to the recipient cell and look at the end of this process both the donor and the recipient have a copy of the F Factor plasmid and so both cells are now considered f+ and what's really neat if it's the fact factor that is shared this bacterium is now capable of making its own sex pilus and by and it's now capable of conjugating with other bacterium of either the same or different species and this bacterium can now share that F factor with other bacterium isn't that neat so through F Factor conjugation the donor cell shares the fact factor with the recipient cell resulting in two F positive cells all right how is this different from HFR transfer first of all what is an HFR cell HFR stands for high frequency of recombination cell so a cell that is called an HFR cell is a cell where something special has happened look at this I want to show you something sometimes the F plasmid the fertility plasmid can actually insert itself into the hosts circular genome it could imagine if I were to cut and paste this circular plas into the circular chromosome of the host cell now look at this that's what an HFR cell is here in in blue in blue you can see the genome the circular chromosome of the bacterial cell and notice what's happened here the F Factor has integrated itself or recombined itself into the genome it's like cut and paste itself into the Genome of the cell now this is known as an HFR cell and because it possesses the genes necessary for building the pilis it forms the sex pilus the sex pilus conjugates with a adjacent cell notice how this adjacent cell is an F minus cell and look what happens through again rolling Circle replication the DNA is transferred from the donor to the recipient and notice this in this case it's not just a plasmid which is being shared with the recipient but the fact Factor as well as genome IC DNA a partial copy of the donor chromosome this this cell down here is not just getting the information for making the sex pilus but it is also obtaining genetic information genes from the donor as well which is really neat then when conjugation breaks the bridge breaks the donated genes are there in the re recipient and sometimes those genes can recombine into the recipient genome and now this bacterium is now called a recombinant it not only has the genes the fertility genes for making its own sex pilus but if you look closely it's also picked up some important genes from the donor as well it's a true new strain and again as I mentioned before the F plasmid isn't the only plasmid in town there are R plasmids or resistance plasmids which carry genes for resisting antibiotics or other drugs there are uh virulence plasmids which have genes for toxins on them as well so they can share all kinds of different traits with one another so now let's move on to the next type of horizontal Gene transfer this type is called transformation and it involves capturing naked DNA from this solution the environment during transformation a bacterial cell will accept small fragments of DNA from the surrounding environment so imagine taking DNA out of the environment and then incorporating that DNA into your own some cells are capable of doing this the cells that are capable of doing this are called competent cells not all cells are able to take DNA out of the environment that happens to be floating around out there and bring it inside only what are known as competent cells these are cells with the correct Machinery to bring doubl stranded DNA inside so let me show you how this might work here on the left you have a bacterial cell which is very damaged and it's dying this cell notice how it circular chromosome has been fragmented the bacterial chromosome is all broken up this cell is dying this cell explodes and lces remember lces lces means to break open this bacterial cell is effectively dead and its inerts have poured out okay the double strand d has poured out now if this bacterium this is the recipient healthy cell if it's a competent bacterium it's capable of taking double strand DNA in from the environment see the double strand DNA is being taken in by this bacterium and if that double strand DNA is incorporated or inserted into the Genome of this cell then a recombinant is formed see this portion in light blue this portion of the recipient is is uh from the host cell from the donor cell from the donor cell and so this is a recombinant and this form of transformation this form of horizontal Gene transfer is called transformation notice how no sex pilus was required a cell died and released double stranded DNA and the competent cell took up that double strand DNA and Incorporated it into its own DNA that's a different way of horizontal Gene transfer now the final form of horizontal Gene transfer is called transduction and what's interesting about this form of Gene transfer is that it requires viruses specifically bacterio phase remember what bacteriophage are they are viruses that specifically infect bacteria and sometimes they could accidentally pick up donor DNA they could pick up some DNA from the donor cell and they could deliver that DNA to a recipient cell so let me show you how this might work this is a generalized form of transduction we're not going to go into uh specialized transduction in this chapter but generalized transduction works like this let me show you how this might work first a bacterio phage remember bacteriophage look like this this is a T4 Fage it has uh this shape like a lunar lander like a like a moonlander this this bacterial phage attaches to the bacterial cell and it injects its DNA its double strand DNA into this bacterium this bacterium is now infected with virus DNA at this point the viral DNA takes over the host cell and it produces more viral components so here the host cell is now producing more viral DNA it's producing viral proteins but notice what's happening here this cell is becoming sick this host cell is now infected and sick and that means that it's circularized chromosome might fragment so here you have a piece of the host's circular chromosome which has broken off it's a separated piece of host DNA and the next step for the virus is really to package and produce new varion right varion are individual viruses and what should the virus package inside look at this red swiggle this red stuff represents viral DNA when new viruses are formed inside of this host cell packaged inside should be viral DNA but look at this guy on the right look at this guy he's accidentally going to package some of that separated pie P of host DNA see so viruses should package viral DNA inside but this guy accidentally packages host DNA inside now when it infects the next cell watch this when this cell breaks open this cell lices or breaks open these Fage are free to go infect the next donor cell right the next cell these Fage are free to go and infect the next cell and notice viruses should deliver viral DNA to bacterium but notice this is this virus delivering viral DNA to this bacterium or is this virus delivering genomic DNA from the previous donor owner it's delivering the wrong cargo it's not infecting the cell with viral DNA it's in it's infecting this cell with DNA from the previous donor by accident this DNA might incorporate itself into this new bacterium and now a recombinant is formed you see how this could lead to recombination because this virus inadvertently introduced genomic DNA from the previous donor and not true viral DNA and that led to a Rec combination event isn't that neat and for the last part of this chapter let's talk briefly about mutations and how they might affect microorganisms mutations are any change to DNA any change to the nucleotide sequence in the genome so anytime a g gets mutated to a c or a t gets mutated to a g anytime there is a change in the genetic code in the genome this is known as a mutation mutation is a driving force of evolution the normal Gene as it is normally found in the population is called the wild type Gene however a mutated Gene once a a gene is altered it's known as a mutant Gene and that forms a mutant strain of bacterium the mutant strain is an organism that bears a mutation mutations can change the function of the proteins that result from those genes what are some causes of mutations mutations can be caused spontaneously these are known as spontaneous mutations and this occurs by random errors in DNA replication so for instance when a cell wants to divide remember the first thing the cell needs to do is copy its chromosomes right now imagine during copying of the DNA mistakes can occur these aren't very common because DNA polymerase has proofreading ability however mistakes can occur so just spontaneously there is a chance that genes obtain mutations they incur mutations when the chromosomes are being copied before the cell divides and this is known as a spontaneous mutation there's nothing we can do about that mistakes are made sometimes however induced mutations as the name suggests result from exposure to either physical or chemical agents that damage the DNA these are known as mutagens and often they're also considered carcinogens these are factors that cause mutations they induce mutations and these mutations Compound on top of the spontaneous mutations here are a list of common chemicals that are mutagens in several labs we work with ethidium bromide which is used to visualize DNA in agarose gels however physical factors such as radiation can also cause mutations as well UV light for instance can cause mutations by causing cross links or dimerization between adjacent perimeters for example thyine thyine diers or cytosine cytosine diers while ionizing radiation for example gamma rays or xrays these can cause double stranded breaks in DNA leading to mutation as well now what's neat is DNA damage can be repaired by a process known as photoactivation photoactivation requires this enzyme called DNA photo lias an enzyme which uses visible light to repair DNA damage caused by UV so you remember those thyine thyine diers we talked about or those cytosine cytosine diers well this enzyme DNA photo lias uses light in order to repair those damaged areas isn't that neat so now let's talk about how microorganisms might be used to determine whether a chemical is a carcinogen or not or whether it's a mutagen or not the Ames test is a great test a microbiology Laboratory test that you can use to determine whether a chemical is carcinogenic and it works based off of this principle of spontaneous mutations versus induced mutations and this is the way the assay works this assay typically uses the bacterium salmonella tyum because of the back mutation potential to synthesize the amino acid histadine what does that mean hold on so what this means is they have found a strain of salmonella that cannot make histamine cannot make histadine because it has a mutation in the histadine gene so again we are starting with a strain of salmonella which is called his minus or histadine minus because this strain of salmonella cannot grow on histadine however a single point mutation can convert this bacterium back to histadine positive so this is a great species or strain this is a great strain to study for this assay here is how it works we take our salmonella which is histadine minus it has a mutation in the gene that codes for the enzyme that produces histadine I should say you place these his minus genes and you spread them on a plate this is a minimal medium which lacks the amino acid histadine now bacterium should not grow on this plate remember because they are not able to produce their own histadine due to that mutation and this plate lacks histadine so bacteria should not grow on this plate unless they incur a mutation now some bacteria do end up growing on this plate and that's those are known as the back mutations they're the ones that fixed the histadine mutation and are now his positive and notice this is your control plate where no uh mutagen or carcinogen was added to the plate so these are what's known as spontaneous back mutations remember there's a certain level of spontaneous mutation that occurs and that's unavoidable so this represents your background level of spontaneous mutation now we want to do our test plate the test plate is similar to the control plate in that it has a minimal medium W which lacks histadine but it also has the test chemical mixed in the potential syst suspected carcinogen or potential carcinogen mixed into the aggar as well again no bacterium should grow however in this case notice there are a huge number of back mutations there are many histadine positive colonies that formed much more than your control spontaneous mutation plate on the left if this number is greater on the right than the left this shows that these were caused by induced mutations so this is truly a mutagen this chemical that you're testing is a mutagen if however this plate on the right exhibited the same number of back mutations as the plate on the left that would have suggested that this is not a carcinogen that there's no more colonies on the test plate than the spontaneous colony on the control plate so that would be labeled a non-mutagen or a non-carcinogen I hope this made sense the as test is really neat so if you've ever wondered how do scientists figure out if a chemical is a carcinogen or mutagen or not well this is one very important way and with that that leads to the end of chapter 6 thank you so much for joining us um let's go ahead and move on to the next chapter please let me know in the comment box below if you have any comments or questions and I'll be glad to answer we'll see you guys for the next chapter Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D Dr D