this is the lecture for chapter eight microbial genetics part two in the first part of the lecture i talked about gene expression and this process is considered the central dogma of biology and it is used to explain how information is stored in dna and used to produce proteins and that those proteins will give the cells their different characteristics the two stages of gene expression are transcription and translation and if you remember transcription is where the information that is stored in the dna is copied into rna producing the mrna messenger rna and then that mrna is used to produce the protein in eukaryotic cells gene expression can be regulated in many different ways and this diagram goes through some of the different examples of where this gene expression can be regulated and it can be regulated in several different ways in the nucleus and that involves the chromatin the form of the chromatin which can regulate whether the dna can actually be transcribed or not and then another big part of eukaryotic gene regulation is rna processing with splicing and adding different components to the messenger rna to allow it to be transported out of the nucleus and into the cytoplasm and even once it is in the cytoplasm there are different ways to regulate protein production so that could involve the degradation of the mrna whether it's intact or not different levels of translational control and then control once the protein is made prokaryotes also need to be able to regulate their gene expression but compared to eukaryotes prokaryotic gene regulation is very simple essentially there's only one way that prokaryotes regulate their gene expression and that is with using an operon and an operon is a sequence of dna that regulates gene expression at the transcriptional level so it is regulating whether transcription is going to occur or not so in other words it's regulating whether you get that mrna produced or not and like i mentioned before an operon is a sequence of dna so this is the example of an operon when we refer to dna in terms of direction we refer to upstream of a dna molecule and downstream of a dna so molecule an operon upstream of the operon so this is the operon here but upstream of the operon is a regulatory gene and this is a gene so every time i say the word gene that means there is a protein product and the protein product of the regulatory gene is the repressor so the repressor is a protein that is produced by the regulatory gene then as you move downstream from the regulatory gene the first part of the operon you come in contact with is the promoter and remember the promoter signals the beginning of a gene so this is where the rna polymerase will bind and start transcribing then downstream of the promoter is the operator and the operator is the sequence of dna where the repressor will bind so that is the purpose of the operator downstream of the operator you run into the structural genes and these are the genes that will actually be transcribed and then translated into the actual proteins which most often are a type of enzyme and so they will catalyze some sort of reaction and then downstream of the structural genes you'll have the terminator and remember the terminator signals the end of a gene so a gene is all of the dna between the promoter and the terminator so this is the structure of an operon operon starts with upstream a regulatory gene that produces the repressor and that repressor is a protein that binds to the operator then downstream of the regulatory gene you have the promoter promoter is the sequence of dna that the rna polymerase binds to downstream of that is the operator again where the repressor binds downstream of that you have the structural genes and downstream of that the terminator so the way an operon works is that if the repressor is bound to the operator then the rna polymerase is unable to transcribe the genes this is sort of like a roadblock if you want gene expression then all you have to do is remove the repressor and then the rna polymerase can go ahead and transcribe the genes and then that mrna gets translated and then you get the proteins so it's a very simple process if you want gene expression you have to remove the repressor from the operator if you don't want gene expression you want to put that repressor on the operator to stop transcription in terms of function there are two types of operons inducible operons and repressible operons and the inducible operon is one i will go through first and the name tells you what it means if it is inducible that means you have the ability to induce gene expression so what that means is that the normal situation means there is no gene expression so this is an example of what the normal situation would be and the normal situation would be that the repressor is active so when we say you have an active repressor or the repressor is active that means the repressor is bound to the operator and because the repressor is bound to the operator the rna polymerase this is the rna polymerase is unable to slide along the dna so it is unable to transcribe those genes so since there is no transcription there's no translation no proteins are produced and again we're going to assume that most of these proteins are enzymes so that means none of these enzymes are produced so you're not getting the biochemical reactions but because this is an inducible operon that means you the cell does have the ability to have those genes expressed and in order to do that you need to have an inducer so the inducer is a molecule that will bind to the repressor and change the shape of the repressor when it changes the shape of the repressor the repressor can no longer bind to the operator and now the rna polymerase is free to slide along the dna and transcribe the genes which will be translated into proteins which means that the enzymes will be produced and you will have the biochemical reaction in terms of function there are two types of operons inducible operons and repressible operons and the first operon that i'm going to go through is an inducible operon an inducible tells you what type of operon this is inducible means it has the ability to be induced that means under normal circumstances this operon has no gene expression but the cell has the ability to induce gene expression and the classic example of an inducible operon is the lac operon and lac stands for lactose so this is an operon where the structural genes encode three genes that produce enzymes that break down lactose and under normal circumstances lactose is absent and so the repressor is in the active form which means the operon is turned off it is not transcriptionally active so in a normal situation the repressor which is produced by the regulatory genes is in the active form so because it is active it binds to the operator and because it binds to the operator the rna polymerase cannot transcribe the genes so no messenger rna is made no enzymes are made and that's fine because there's no lactose to be broken down but if the cell does run into lactose the situation will change so when lactose is present the lactose will be converted into a similar form called aloe lactose and this aloe lactose serves as an inducer and if an inducer is going to bind to the repressor and change the shape of the repressor and that inactivates the repressor so because this repressor is inactivated it cannot bind to the operator so because the repressor is not bound to the operator that means the rna polymerase can slide along the dna it can transcribe those three genes and they get translated into the three types of enzymes that break down lactose so now the cell has these three enzymes to break down lactose and it can use lactose as a food source a nutrient source once all the lactose has been eliminated and there is no more lactose present so the lactose is absent it goes back to the having a repressor that is activated because all the lactose is gone so it goes back to active form and turns the operon off stops gene expression and this is very efficient because the bacterial cell does not need to make enzymes to break down a nutrient it does not have the second type of operon in terms of function are repressible operons and like the inducible operon if you look at the name it describes what is happening so a repressible operon means it's an operon that has the ability to repress gene expression so if it has the ability to repress gene expression that means the normal situation is that gene expression is occurring and those enzymes are present so in this case when the repressor is produced by those regulatory genes it is in the inactive form so because it's in the inactive form it is not bound to the operator so the rna polymerase is free to slide along the dna and transcribe those genes so those protein products of the gene are being produced but if for some reason too many of those enzymes are being produced the cell has the ability to turn off gene expression and to do this the cell needs a co-repressor so co-repressor like coordinate co-worker the co-repressor will bind to the repressor converting it into the active form and the repressor will bind to the operator blocking the rna polymerase from transcribing these genes the classic example of a repressible operon is the tryptophan operon trip operon and tryptophan is a type of amino acid that is used in making proteins and in the tryptophan operon the genes the structural genes are a series of proteins that encode enzymes that synthesize tryptophan so most bacterial cells need a constant source of tryptophan in order to make the proteins so for this repressible operon tryptophan is absent or in very low concentrations so the repressor is in the inactive form so when that repressor is made from the regulatory genes it is automatically made in the inactive form so because it is inactive it does not bind to the operator so the rna polymerase is free to transcribe those genes into mrna then they get translated into proteins and those proteins again are the enzymes that are synthesizing tryptophan if for whatever reason too much tryptophan is present so the cell is making too much tryptophan more tryptophan than it needs to use then it's not efficient for the cell to keep making even more tryptophan so what would be efficient is to shut down this gene expression so in order to do that the tryptophan that is present is going to act as a co-repressor so the tryptophan will bind to the repressor and convert it into the active form so now you have an active repressor which will bind to the operator and will block the rna polymerase from transcribing the genes so in this case with the co-repressor it's going to make the repressor active which will turn the operon off which will stop gene expression the last topic i want to talk about in this lecture is genetic change in bacteria gene expression is also referred to as the central dogma of biology because it's a very important concept in biology in general and in microbiology specifically and i've spent a lot of time going through the mechanics of it talking about transcription and translation but i want you to remember the overall importance of gene expression and remember the overall importance is that we start with dna and again the molecule of information and specifically the little packets of dna information that we're interested in are the genes and every time i refer to the word gene think of that as a segment of dna that encodes for information to form a protein and most of those proteins that we are talking about are some sort of enzyme and an enzyme is a protein catalyst that catalyzes some sort of reaction and depending on the type of reactions that a cell can perform that gives the cell different characteristics and this is very important in biology in general but it's also very important in microbiology in the next chapter i'll be talking about biotech and introducing different genes into different types of bacterial cells allows us to produce different protein products and there are many types of proteins that we use microbes to produce things like human growth hormone and vaccines so we can take a gene from another organism like a human or a virus insert that gene into the bacterial chromosome and the bacteria will produce the protein so that's in important in terms of biotech but also in terms of medicine this process is very important sometimes bacteria acquire new genes which gives them a new unique protein and sometimes those proteins are enzymes that can break down antibiotics such as penicillin and so if a bacterium acquires a new gene that gives it a new ability to break down penicillin then that bacterium is considered antibiotic resistant so it's very important for us to understand all the different ways that bacteria can acquire new genes because ultimately it's going to give them new characteristics there are four different ways that bacteria can acquire new genetic material the first is by mutation the second is by transformation the third is by conjugation and the fourth is by transduction and remember the importance of acquiring new genetic material is because it will give the bacteria new characteristics the first type of genetic change i'm going to go through is mutation but before i do that i just want to remind you about protein structure you should have learned before that the structure of a protein is very important to its function and if you change the structure of the protein in any way you are going to alter the function and protein structure is fairly complicated there are four different levels the primary level of structure is the sequence of the amino acids in the chain in the polypeptide chain secondary structure involves how that initial change starts to fold on itself either in an alpha helix or a beta pleated sheet and that all relates to the formation of hydrogen bonds between the amino acids then tertiary structure is the three-dimensional structure of this polypeptide how it all folds in on itself and again that is uh facilitated by hydrogen bonding and then the last level of structure is quaternary structure how this one protein interacts with other proteins to form a functional enzyme so the sequence of amino acids is very important to the ultimate final structure and functionality of the protein when referring to mutation we're referring to a change in the sequence of dna and mutations can be caused in different ways they can be caused by things outside of the cell like chemicals or radiation or they can be caused by mistakes that the enzymes make during dna replication so mistakes that the dna polymerase will make during replication but however a mutation is caused a mutation is basically a change in the sequence of dna and this diagram goes over one type of mutation and so this part shows you the normal situation so the blue is the dna double-stranded dna and this is the template strand here so for gene expression this template dna would be transcribed into an mrna and this gives the complementary mrna sequence then this mrna would be translated into the protein so this is the normal protein and the assumption with uh protein production bacteria is that every protein they produce is essential to their function so if you eliminate that protein or change that protein in any way the cell will die so one example of a mutation is a base substitution so this is where you're substituting one base for another so the normal sequence was g at this location and now an a has been substituted instead of the g so that's the mutation and base substitution you have substituted one base for another and as this template dna is transcribed you get a change in the mrna but when you actually go through and translate it there is no change in the amino acid so this is considered a silent mutation because there is no effect in the amino acid sequence the bacterial cell can produce exactly the same protein and that protein can function exactly the same way so the cell is completely unaffected so this is considered a silent mutation here is another example of a base substitution mutation at a different location so this is the same setup as before and in this case this cytosine is going to be mutated to a thymine so the original wild type wild type means the normal type sequence was a cytosine at that location now it is the thymine and as this template dna is transcribed it will produce a change in the mrna where this is now an adenine and when this is translated there will actually actually be a change in the amino acids so instead of glycine it is now changed to serine and this type of mutation is considered a missense missense is when you have a change in one amino acid now the effect of on the bacterium we wouldn't know unless we actually performed this and determined if this enzyme is still functional or not because depending on the location of this amino acid if it is an essential amino acid for protein structure then that means the protein will change its structure which means its function will change and most likely when an enzyme has a change in structure it becomes non-functional so if this enzyme is no longer functional the most likely effect is that it will be a lethal effect on the bacterium the bacterium will not be able to survive but on the other hand if this amino acid is not an important amino acid for protein structure then the bacterium may still have a functional protein and this mutation will not have an effect on the function of the bacterium another example of a base substitution mutation is here where an adenine has been substituted instead of a thymine instead of the original thymine and again it causes a change in the mrna and now what this does is it creates an early stop codon so for this type of mutation it's referred to as a nonsense if you generate an early stop and in this case your protein is not produced at all and this would be a lethal effect on the bacterium because again we're working under the assumption that all proteins produced in a bacteria are essential for the function of the bacteria so this protein is no longer able to be formed so it's most likely going to have a very negative effect a lethal effect on the bacterium this is a different type of mutation this is a change in the number of nucleotides that are making the sequence of the dna so in this case the original wild type gene had three adenines in a row and this mutation one of those adenines has been removed so in this case what will happen is you will get a frame shift mutation so in reading those codons remember you start at aug and then you have aag uu ggc and so on what will happen is you will change that reading frame so originally before the mutation will be the same aug agg or aag and now it will be uug and then g c u and so on so what this will do is it will change the sequence sequence of amino acids from where that mutation occurred and this is called extensive missense so from the point where that mutation occurred you're going to have different amino acids and this will completely change the protein product which means it won't have the original function which could result in a lethal effect on the bacterium or it's possible that this new protein has some sort of new function that gives the bacterium a new characteristic and enhances it in some way so even though mutation is not a big source of positive effects on bacteria it is possible that some mutations may actually give a bacterium a new characteristic so now i have some practice problems for you in terms of gene expression and mutation so a question could be what would happen to the protein product if the third c was mutated to a t and that refers to the sequence so the first thing you will have to do is perform gene expression to see what the normal protein is and again you can determine that this is dna because it has thymine in it so the first thing that you would have to do is transcribe this into rnas use complementary base pairing so this g would pair with a c a would pair with u remember you're transcribing into rna then g then a u g u c g a g g c g u u a a g g and of course you need to determine what the protein product is so for the protein product you're going to have to translate and remember the first thing you want to do is find your start codon the start codon is the aug so you slide along your mrna until you find your aug in the set your reading frame and then your codons are every three nucleotides you see g is a codon agg is a codon c g u is a codon u a a is a codon and so on and then you use the genetic code to identify your protein and the aug is the start codon and often that involves the amino acid methionine then ucg you see g is serine serine and agg a g g is arginine and c g u c g u is arginine and uaa uaa is the stop so then you stop so the normal protein the wild type protein is methionine serine arginine arginine and the question is what would happen to the protein product if the third c was mutated to a t so third c one two three and if this was mutated to a t so it is no longer a c it's a thymine then this nucleotide in the rna would be an adenine and now that has changed that codon so now it's uca so it's u c a is serine so the new amino acid would be serine which is the same which means the protein would be the same and this would be a silent mutation another practice problem using the same sequence what would happen to the protein product if the third a was mutated to a c and again finding the normal protein complementary base pairing into rna so this would be c u g a u g u c g a g g c g u u a a g g and again we looked for that start codon you always find your start code on and then every three nucleotides is your codons that you're looking for and again when we translated this this was methionine start codon is always methionine and then serine and then arginine arginine and stop stop codon all right now for this mutation what would happen to the protein product if the third a was mutated to a c third a one two three so if this was mutated to a cytosine then this would be a guanine and now the new codon is gcg so g c g this would become an alanine so now it would be methionine alanine arginine arginine and this would be a missense because you have had a change in one amino acid and it's possible that this uh protein would still be functional or it could be a lethal this could be an important amino acid and it could be lethal or it could be beneficial so actually maybe this makes the enzyme even better so you don't know what the effect would be on the organism with a mist sense the last practice problem with the sequence what would happen to the protein product if the second g was missing so for this uh first transcribe into rna so c u g a u g u c g a g g c g u u a a c c so you get it transcribed into rna then you have to identify the protein product so you always look for that start codon aug here is the aug and then every three nucleotides is a codon you translate so this is the methionine again the star codon is always methionine and then there is the serine arginine arginine and stop codon all right now for this one what would happen to the protein product if the second g was missing so here's the first g here's the second g so now that second g is missing so the rna would not have that second c and this would change your reading frame so now instead of the first codon after the start codon being ucg it's going to be uga and then ggc and g u u and aac and this is completely going to change your protein because the start codon is the same methionine it's methionine then the second one will be uga so u g a is a stop so this is an early stop so this would be a nonsense mutation nonsense mutation in a nonsense mutation will definitely have a lethal effect on the bacteria transformation is the second way that bacteria can acquire new genetic material and the basic definition for transformation is when a bacterium picks up naked dna from the environment and dna is not normally found in the environment the only way you really get naturally get dna into the environment is if a cell dies and lyses and releases fragments of its chromosome into the environment and some bacteria have the ability to bind to these fragments of dna that they find in the environment and to transport them into its cytoplasm once the dna is transported into the cytoplasm sometimes it can splice into the chromosome and whatever proteins are encoded by this fragment of dna now this bacterium has the ability to produce those proteins so this bacterium has acquired a new gene from that original bacterium and now has the ability to produ produce some sort of new protein which will give it a new characteristic the third way that a bacterium can acquire new genetic material is by conjugation and i've already mentioned conjugation i talked about this when we were talking about the appendages of bacterial cells and i talked about pili and the singularis pilus so conjugation is defined as when a plasmid is transferred from one bacterium to another by a pilus and this occurs between f positive cells f positive f stands for fertility factor which means that the bacterial cell contains the plasmid and is able to form a pilus so an f positive cell can conjugate with an f negative cell one that does not have a plasmid and during conjugation you get the pillars forming a bridge between the two cells once that is formed then the plasmid can replicate a copy and transfer a copy of that plasmid over to the f negative cell converting the f negative cell into an f positive cell and this plasmid has been transferred to this bacterium so this is new genetic material it never had before and often genes you find on plasmids are genes for toxin production and antibiotic resistance so often acquiring a plasmid will make a bacterium more pathogenic because it will probably get genes for toxin production and or antibiotic resistance the last method by which a bacterial cell can acquire new genetic material is by transduction and transduction is basically defined as when bacterial genes are passed from one bacterium to another by a phage and there are two types of transduction i talked about during the virus chapter there's generalized transduction and specialized transduction and generalized transduction if you remember occurs when a phage infects the bacterium and during penetration the bacteria's chromosome the bacterial chromosome is broken down degraded into different fragments and during maturation one of those fragments is accidentally packaged into a capsid so upon release this phage particle now contains bacterial genes so when it attaches and penetrates into the next host cell it is actually injecting bacterial dna and that bacterial dna can then splice into the host cells chromosome and now whatever genes are on that fragment of bacterial dna now that cell can produce those proteins specialized transduction also involves bacterial genes being transmitted from one bacterium to another by a phage but in this case it involves a prophage and remember prophages are formed by lysogenic phages and a prophage is the phage dna that has spliced into the host cells chromosome and it can stay there until that cell gets a trigger like a change in temperature and then that prophage will splice out but in this case it takes along a bacterial gene with it so when that phage then goes through biosynthesis and maturation in all the capsids including the phage dna there's also this gene from the original bacterium so when this new phage attaches and penetrates into the next host it's going to inject the phage dna which also has this bacterial gene so when the next prophage is formed it's not only the phage dna but also that bacterial gene from the original host and so now this bacterial cell has acquired the gene from the original host so again the phage is transmitting bacterial genes from one bacterium to another the importance of genetic change in bacteria especially in terms of medicine is the development of antibiotic resistance and genetic change in bacteria can occur in four different ways first you could have mutation in the genome that could lead just randomly to an antibiotic resistance gene or you can have transformation where the bacterium is picking up naked dna and bringing it in you could have conjugation where a plasmid is being transferred from one bacterium to another or you can have transduction where those resistance genes are being transferred by phage but either way it is very easy for bacteria to exchange genes between each other and that is how the spread of antibiotic resistance occurs and we will be talking about this again when i go through the chapter on antibiotics