i'm dr d dr d dr d dr d dr d dr d dr d explain stuff hey everyone dr d here and in this video we are going to be covering chapter 10 from our genetics essentials uh fourth edition textbook from dna to protein transcription and rna processing so this chapter when you're dealing with transcription you're dealing with rna and it's hypothesized and evidence suggests that rna was the original genetic material believe it or not uh the you know people have always wondered where did life come from and the best evidence suggests that dna was not the original carrier of genomic information of heritable information it was rna why because ribozymes rna enzymes are possible and in the lab setting you can actually get rna short stretches of rna that are able to catalyze themselves so you can have rna creating more rna in the lab and that's essential to propagation of information is the ability to copy yourself right right now in a cell you have genomic dna right you have the genomic information in the form of dna and that dna is propagated and copied by proteins correct not just proteins but rrna trna mrna the mrna and the and the proteins help copy the dna so the thing is how did dna get copied before a cell existed before proteins existed well the idea is the earliest genomic information was carried as rna why because rna has a ribozyme or enzymatic ability it can copy itself it's been shown in labs that rna can copy itself and then because rna is less stable than dna dna formed as the source of the genomic uh information and rna assisted in the copying of the dna and proteins came later on or at least that's the hypothesis isn't that very fascinating that that is what we think how life came about rna was the heritable material rna ribozymes copied themselves and then later on as a more stable carrier of genomic information dna came about and proteins so with that you know let's see how evidence builds towards that towards that idea but with that let's move on with our study of transcription this is the first part of gene expression this is where genomic information is copied into mrna right transcription is where a gene is com is copied into complementary mrna and before we get started with transcription let's talk about a little bit about rna itself the primary structure the secondary structure of rna so here's a strand of rna you can see that a strand of rna is directional meaning it has a five prime end with a phosphate group much like dna has a five prime n with a phosphate group a strand of rna also has a three prime end with a with a hydroxyl group on the three prime carbon of the ribose sugar much like dna you have a sugar phosphate sugar phosphate backbone right and it's this straight backbone and the difference here obviously is that you have a hydroxyl group on the two prime carbon of the ribose sugars here you see you have a hydroxyl group whereas in dna you're missing that oxygen that's why it's called deoxy ribose and another difference here with rna is that you have uracil uracil instead of thymine so you have a's g's c's and u's u's instead of t's but do recall that u's still pair with a's just like t's pair with a's in rna u's pair with a's with two hydrogen bonds so this is called the primary structure of rna the primary structure is essentially the list of nucleotides in order but keep in mind that base pairing can still occur with mrna or any rna that is it doesn't have to be mrna trnas fold as well and many other rnas fold in on themselves recall that rna is single stranded usually single stranded so the types of double the types of secondary structures you can form are when rna loops back on itself so look at the secondary structure here are this rna strand is looping back on itself and this is called a hair pin so you form a hair pin loop back and then there's another little loop here with another hair pin you see so secondary structures are usually they and they entail a strand of rna looping back on itself uh you know and then complementary base pairing with itself forming hairpin structures uh that's essentially what the secondary structure of rna looks like so again here's a handy dandy table showing you the structures of dna and rna and comparing the two we've already addressed all of this but please touch on that now i bet you didn't know um that there are so many different kinds of rna in the cell uh you've heard of mrna we've talked a lot about mrna the messenger rna you can find this in bacteria and eukaryotes and archaea this is the copy of the gene mrna is the copy of the gene but you also have rrna remember the ribosomal um the the rna that's part of the ribosome trna which assists in uh translation uh you can have and we'll touch on some of these later on but small nuclear rnas small nucleolar rnas micro rnas small interfering rnas pee wee interacting rna non long non-coding rna crispr rna there's many different types of rna in the cell each with a different function some of those functions not known just like the long non-coding rna is still a big mystery in genetics today so again you have all these different classes of rna uh but they all share one thing in common they're made up of uh the ribo nucleotides a's g's c's and u's you know and then they can form the secondary structures so they have different jobs and some of them again are known as ribozymes some of these can serve as enzymes when you think enzymes in biology most of you think of proteins right you know there's uh there's this protein or that protein and and these proteins are enzymes in the cell but you should also understand that there's another whole class of enzymes called the ribozymes and these are enzymatic rna and sometimes the rna and protein work together to form an enzyme so for example the the ribosomes themselves are a ribosome the the ribosomes themselves are ribozyme because part of the ribosome is protein and part of the ribosome is rna does that make sense the um during splicing the spliceosome proteins during rna splicing that's a ribosome so some some of these ribozymes are enzymatic rna and that rna itself is enzymatic some of these ribozymes it's a hybrid molecule of rna and protein and that's an enzyme so the so the the enzyme wouldn't work without its rna component if that makes any sense so let's look at concept check number one which class of rna is correctly paired with its function well let's look small nuclear well we didn't go through that table right but i will tell you that here transfer rna trna this is the one that attaches to an amino acid remember uh we're going to talk about this in the next chapter actually the next chapter chapter 11 we're going to talk about how transfer rnas are these small rnas that attach to amino acids and they facilitate translation translation during gene expression so you may want to go through this table and learn the various functions of the different types of rna at least know the different the different functions we're going to touch on this this one crispr rna which is exciting new field that only in the last 10 years or so has come about but it's an exciting new field of genetic engineering and genetic research where we can edit genomes using this kind of technique so again i told you there are different forms of rna some are exclusive to eukaryotes some are exclusive to prokaryotes you know crispr is found in only prokaryotes the others are found in uh some are found in only eukaryotes and some are found in both and don't forget the central dogma of uh molecular biology remember the concept of the set central dogma of molecular biology information flows from dna to rna to protein and this is known as gene expression right uh the first part of gene expression is called transcription that's what we're going to be talking about in this chapter where dna is copied into rna and then the second part of gene expression is called translation and that's where the rna message mrna message is read to protein and that's what we'll be discussing in the next chapter and then there there is an exception here that there are some rna viruses that can copy rna directly from rna but those are the exception there so so you and i our cells do not have the ability to copy rna to rna um only and only enzymes from viruses uh called rna-dependent rna polymerases only those enzymes are able to copy rna to rna oh and the and uh some some enzymes from viruses can even copy rna to dna like the retroviruses hiv for example but you and i we do not possess the enzymes capable of copying rna to rna nor do we possess the enzymes capable of copying rna to dna we only have the ability to copy dna to dna that's during s phase of mitosis where you copy the chromosomes and we can copy dna to rna can't we that's the process of transcription for instance so here you can see under the electron microscope dna molecules undergoing transcription so these little christmas tree structures are essentially dna down the middle you see the the main branch here is dna and then these uh these or the trunk the trunk down the middle is dna and then the branches off the sides are the rna so this would be for instance a gene right this is a gene and transcription would start somewhere over here and you're copying the gene and as you copy the gene from right to left uh the tail of the mrna becomes longer and longer and longer right so it forms these christmas tree-like structures so here it is with an actual electron micrograph of the christmas tree-like structures and then in b uh the trunk of each christmas tree is called the transcription unit which is dna a dna molecule a gene right and then the branches are the mrna the mrna complementary copy of that gene notice how the mrna is short here the branches are short here that's because you've only copied this much of the dna but by the time you're here you've copied all of the dna so the christmas tree is going to be longer the branches are going to be longer because you've copied more of the dna as you travel from right to left as you copy from right to left so here in figure 10.4 you see rna molecules are synthesized that are complementary and antiparallel to one of the two nucleotide strands of dna the template strand here's the dna double strand dna this is double strand dna in your body one of these is here's the gene on the right right here this is the gene right this is the gene what you need to know is that what you're doing to the double strand dna chromosome your uh unwinding it right so an enzyme called rna polymerase unwinds the dna and then it copies only one strand you see the bottom strand is being copied and that's called the template strand and you're reading the template strand three to five because the mrna is being built five to three just like dna is built five to three mrna is built five to three as well so you see what's happening the rna polymerase is building on the three prime end of the newly synthesized mrna so right here if you see a g you're going to put a c if you see a c you're going to put a g if you see it a that's a trick one would it what happens with rna polymerase if it sees an a on the template strand of the dna it's going to put a u if it sees a t it'll put an a right so let's read what it says here about this figure uh part one rna synthesis is complementary and antiparallel to the template strand again complementary yeah because you're complementing the dna and antiparallel the rna is being built five to three but it's reading three to five part two new nucleotides are added to the three prime o h group of the growing rna so you see just like dna is only built in a five prime to three prime direction rna is also only built in a five prime to three prime direction as well okay so you see mrna is being built five prime to three prime the non-template strand is not usually transcribed so what's happening on the top part of this what's called transcription bubble the top strand is not being copied only the bottom strand is being copied okay now we're going to talk a little bit about the template strand that bottom strand the it's the transcribed strand it's called the template strand and part of that template strand is called the transcription unit what's being transcribed what's being copied into rna well in front of the transcription unit in front of what's being transcribed you have a stretch of dna called the promoter this is where the r rna polymerase binds we're going to talk about this in a minute the promoter is a stretch of dna in front of the gene upstream of the gene they call it where the rna polymerase binds then you've got the rna coding sequence downstream of the promoter that's the sequence that's going to be copied into complementary mrna and then you need to know where to stop copying the gene and that's called the termination site or the terminator so let's look at a gene here you have dna chromosome part of a chromosome double strand dna you have a gene a a gene b and a gene c take a look uh in this example you could see that the that g a and g and b and g and c are going to be transcribed into mrna and ultimately into protein but look at this with uh gene b the bottom strand is the template strand but with gene a and g and c the top strand is the template strand so this should tell you something important and that is that transcription does not always occur on the same dna strand if you have a chromosome some genes are you know the template strand is uh the the top strand and some genes the template strand can be the bottom strand and then look at these arrows as well you see how this arrow is going to the left but these two arrows are going to the right that means that transcription proceeds to the right with genes a and c but it proceeds to the left with gene b so even the direction of transcription is not uniform along the chromosome so let's do concept check number two what is the difference between the template strand and the non-template strand well isn't the template strand transcribed and used as a source of of complementary dna so you complement the template strand and you build on it in a five prime to three prime direction and in an antiparallel direction to form the mrna the non-template strand is not actually copied by the rna polymerase so again the template strand is the dna strand that is transcribed into an rna molecule a complementary rna molecule the non-template strand does not transcribed here we can see in figure 4.0 sorry 10.6 a transcription unit includes a promoter and remember what i said a promoter is it's a stretch of dna upstream of the gene of interest where the rna polymerase attaches remember the rna polymerase is the enzyme that's going to create the mrna it's going to copy the dna into rna a region that encodes rna and a terminator so on the on the transcription unit you have three things in each transcription unit a transcription unit includes a promoter it includes the region that encodes rna and the terminator so you can see here here's a gene right double-stranded dna on a chromosome somewhere here's a gene you have the promoter region in yellow you have the rna coding region in lilac and you have the terminal terminator region in burnt orange here okay only the lilac region is going to be actually copied to rna you see that only the only the rna coding region is going to be copied into rna and part of part of the terminator as well not all of the terminator part of the terminator because this is the transcription stop site or the transcription termination site so you see this is your rna transcript the rna transcript does not include the promoter the rna transcript includes the rna coding region and part of the terminator okay does that make sense again what's the function of the promoter the promoter is a region of the gene upstream upstream means you know the away from up from where transcription starts upstream of the the rna coding region where the rna polymerase will bind this is where the rna polymerase will bind but it is not actually dna that's going to be copied into rna the promoter contains what are known as consensus sequences these are dna sequences that recruit the rna polymerase that allow the rna polymerase to attach to the promoter so what do we need for transcription to proceed you need uh the rna polymerase i told you about the rna polymerase you need building blocks remember in dna it's dntps deoxy nucleotide triphosphates in rna you have ribonucleoside triphosphates so our ntps are the building blocks are the nucleotide building blocks of rna dntps are the nucle are the building blocks nucleotide building blocks of dna so what do you need you need the building blocks rntps you need the bacterial rna polymerase if you're talking about transcription of bacteria and in bacteria you also need what's called the sigma factor this is a protein that facilitates binding to the promoter when the transcription starts so rna polymerase requires sigma factor to bind to the promoter to to start or initiate transcription here's what an rntp looks like ribo nucleoside triphosphate or rntp you have a base which is what a g c or u right you have your sugar notice how you have a hydroxyl group on the two prime carbon of this sugar so it's ribose sugar it's the pentose sugar is ribose and then you have three phosphate groups three phosphate groups so it's a triphosphate this is a building block of mrna or any other stretch of rna this is the this is the subunit or substrate i should say for uh rna polymerase rna polymerase requires these uh substrates in order to build rna by linking them together linking these together so you can see here in figure 10.8 in transcription nucleotides are always added to the three prime end of the growing rna molecule just like dna like i said before remember with dna we talked about dna replication how the daughter strand is synthesized in a five prime to three prime direction meaning dntps can only be added to the three prime end of the daughter strand of dna same thing with rna our ntps can only be added to the three prime end of the rna so are so it's not pictured here but rna polymerase is actively synthesizing rna and it's creating the rna in a 5 prime to 3 prime direction which means the bottom strand here is the template dna strand and you're traveling along the template dna strand in an antiparallel fashion so the anti so the template is going three to five you're you're traveling along the template three to five you're building your newly synthesized rna five prime to three prime and what does it say here initiation of rna synthesis does not require a primer unlike dna synthesis rna synthesis does not require a primer you can just start copying the dna into rna immediately from scratch and as you travel along the as you travel along the the gene and you're copying that dna this this bubble moves down the gene this is called the transcription bubble and again it's being formed the reason this unwinding occurred is because what what you can't see is this enzyme called rna polymerase which is traveling along the dna unwinding the dna and facilitating transcription right number two new nucleotides are added to the three prime end of the rna molecule so you keep complementing the dna with rna copying in a five prime to three prime direction then number three dna unwinds at the front of the transcription bubble so you keep unwinding the dna you keep traveling forward you keep track you keep copying the dna into mrna and then behind the transcription bubble the as the rna polymerase travels the the dna rewinds behind so here you can see that much like eukaryotic dna polymerase rna polymerase is just as diverse you have multiple different forms of rna polymerase rna polymerase one two three four and five each one has a different job in the cell some are some are exclusive to plants and not to animals you don't need to know this for the uh scope of this class but just be aware that much like eukaryotic uh dna polymerase and how they remember there were several different forms rna polymerase is the same there are different types of rna polymerase and eukaryotes it's not as simple as in the prokaryotic system and each one has a different job concept check number three time what is the function of the sigma factor during transcription remember i said the sigma factor is required for proper binding of the rna polymerase to the promoter during transcription and that is to initiate or start transcription so again it says here the sigma factor controls the binding of the rna polymerase to the promoter without the sigma factor you're not going to get transcription because the rna polymerase will not be able to attach to the promoter to initiate transcription again during initiation what else is going on the substrate for transcription the substrate are those rntps i told you about remember the ribonucleoside triphosphates or rntps these are yours a's your c's your g's your u's and these are added to the three prime end of the growing rna molecule the growing copy of the dna the transcription apparatus is eukaryotic rna polymerases so you got the the enzyme is rna polymerase the substrate are the rntps and then you copy the template strand of the gene now in bacterial promoters we're kind of flip-flopping between eukaryotic and prokaryotic transcription here but back to prokaryotic bacterial promoters contain what's known as a consensus sequence which is sequences that possess considerable similarity at the minus 10 position on transcription this means 10 bases upstream of where transcription starts and dna is actually copied to rna that's called the transcription start site 10 bases upstream of that is called the 10 minus 10 consensus and this is your prep now box which reads t-a-t-a-a-t also known as the top box the t-a-t-a-a-t serves as a binding site for rna polymerase and 35 bases upstream of the transcription start site you have your another consensus sequence called ttgaca so when you have the tata box 10 10 basis upstream of the transcription site and the ttg aca site you know another 25 bases upstream from that that serves as the recognition site for the rna polymerase to bind that's that's how rna polymerase knows to bind to the promoter that's that's why the promoter works right and what is a consensus sequence it's simply a consensus of actual sequences similar sequences and when you line up these sequences from the promoters of different organisms that's how you gain your ears consensus so the consensus sequence comprises the most commonly encountered nucleotides at each site for for different organisms so here you can see more details about this promoter i told you about this is this is okay this is the the the transcription start site it's called the plus one site that's because that's where the first rrna the first rntp i should say the first rntp is added so the transcription actually begins and you start making your rna at this point here called the transcription start site and you're traveling in this direction copying the template strand the bottom strand of dna into rna right into the rna transcript in a five prime to three prime direction however remember ten base pairs up from that is your prim now box or the tata box and then 25 bases up from that is your ttg aca site so when you have this consensus sequence at minus 35 this is called the minus 35 sequence when you have this tata box at minus 10 that's what allows the promoter to serve as a promoter to recruit rna polymerase in order to begin transcription and remember you don't actually copy the dna until you reach the transcription star site so let's talk a little bit more about initiation the initial rna synthesis occurs no primer is required remember you don't need to lay down a short stretch of rna like you do with dna replication um no rna is required so you no primer is required because you can just start copying the dna right away at the transcription start site the location of the consensus sequence determines the start site so again it's the consensus sign sequence your prep now box and your minus 35 sequence dictate where that plus one site is the transcription start site is and then that's followed by elongation which is rna elongation this is where rna polymerase is actively doing its job it's laying down you know those uh phosphodiester bonds linking the rntps together to form your rna in a five prime to three prime direction that's what elongation uh entails elongation is the active process of you know copying the dna into rna so how does transcriptions transcription work let's go back to the beginning here's your promoter this is the gene you're trying to copy this is the transcription start site which is the plus one site this is your core rna polymerase this is uh basically rna polymerase along with a couple other proteins and known as the core rna polymerase remember the rna polymerase can't bind to the promoter and the minus 10 or minus 35 sequences without the help of sigma factor sigma factor associates with the core enzyme to form what's known as the holoenzyme the holoenzyme is the polymerase plus sigma factor that's the holo enzyme once sigma factor binds to polymerase the two can or the the group actually polymerase is usually more than just one protein this group of proteins can bind to the promoter then the promoter it gets un unraveled um uncoiled right the the dna strands are separated that's when sigma factor uh can usually sigma factor can leave oh but maybe it's after initiation so the template strand is exposed the template strand is exposed and then the holoenzyme moves to the transcription start site it grabs the first r ntp right the first rntp for the transcription start site and you have now initiated transcription so you're going to copy the bottom strand and you're going to start building your new strand of rna 5 prime to 3 prime okay at this point the sigma factor can get removed you see the sigma factor is released as rna polymerase moves beyond the promoter so no longer need the sigma factor and what do you do now it's time for elongation the rna polymerase core enzyme proceeds downstream and it copies it helps to copy the the dna into rna in a five prime to three prime fashion so you can see that new rntps are being added to the three prime the growing three prime end of the rna conclusion rna transcription is initiated when the core rna polymerase binds to the promoter with the help of sigma factor and then sigma factor can pop off and elongation continues so during elongation the molecular structure of the eukaryotic polymerase ii and how it functions during elongation has been revealed through the work of roger kornberg and his colleagues so again it's a little bit more complex than in prokaryotic transcription but a lot of the same mechanisms you know are consistent and then you have termination of transcription so let's look at termination of transcription there's two main forms of termination of transcription you need to know about row dependent termination and row independent termination it all depends on whether or not you use this row factor this row protein or not during uh termination of transcription so let's take a look here at row dependent termination so you have rna polymerase it runs into the terminate terminator site and at the red site you have binding of rho rho binds to the red site and moves toward the three prime end of the newly synthesized rna when rna polymerase encounters a terminator sequence it pauses so rna polymerase pauses allowing rho to catch up when rho catches up using helicase activity rho unwinds the dna rna hybrid and brings an end to transcription so now you get everything separated here the rna separates from the dna and you have uh effectively ended transcription and why it was because the rna polymerase stalled at the terminator sequence allowing row to catch up rho caught up with its helicase activity and ended transcription now during row independent termination this is a slightly different technique this is where you have these inverted repeats in the terminator you have inverted repeat followed by a string of six adenine nucleotides so you see an inverted repeat means that the sequence here is a palindrome is is an inversion of the sequence here and what happens then is that you transcribe those repeats those repeats then because they're they're an inversion they they bind to each other the the repeats bind to one another forming a hairpin right a hairpin which destabilizes the dna rna pairing and then you have a stalling out at this stretch of six or seven a's and that alone allows the rna transcript to separate from the template terminating transcription so conclusion transcription terminates when the inverted repeat forms a hairpin followed by a string of uracils remember uh these these a's and u's have only two hydrogen bonds so two hydrogen bonds is not very strong right compared to a c's and g's which form three hydrogen bonds these a's and u's are only forming two hydrogen bonds along with this destabilizing hair pin that causes the rna polymerase to stall and fall off of the template strand right so again there's two ways of terminating transcription one is using row factor and the other one is by forming a hairpins with these repeats all right before we move on to this concept here of splicing let's go ahead and take a quick break time with gizmo and wicket and we'll come back strong to finish off this chapter what do you say [Music] all right everyone welcome back from break time with gizmo and wicked let's go ahead and keep moving on with this chapter now what i was telling you about was how transcription works but recall that from biology 1406 you should have learned that eukaryotic genes and prokaryotic genes are fundamentally different in one major way and that is that eukaryotic genes possess stretches of dna called exons which code for protein here you can see the ovalbumin gene and how it possesses eight stretches of dna that code for the ovalbumin protein and that's known as the stretches of dna called exon 1 exon 2 exon 3 4 5 6 7 and 8. again exons are stretches of genes that code for the protein that the gene is trying to form and in between those exons you have stretches of dna known as introns introns are non-coding dna so this would be called intron 1 here intron 2 intron 3 intron 4 intron 5 6 and 7. and the problem with eukaryotic transcription is that let's say the promoter is up here somewhere for you know the promoter of transcription rna polymerase would bind up here somewhere rna polymerase would then travel down this way and copy exon 1 exon 2 exon 3 exon 4 but it would also copy the intron information as well does that make sense so after transcription the introns need to be removed by rna splicing this segment of rna needs to be removed in order for the exons to come together so that the ribosome can then read the exon information only off of the mrna to form the correct protein if you don't splice out the introns you're not going to end up with the correct protein and the protein may not function at all so here same thing with cytochrome b gene you've got exons one two three four and five you also have intron information between after transcription you have to remove the intron information from the transcript from the rna transcript in order to have your mature mrna ready for translation recall though in prokaryotes there are no introns so in prokaryotes the entire thing is a giant exon there's one giant exon does that make sense so in prokaryotes there is no need for rna splicing because there are no introns and exons to deal with the entire gene is an exon now don't forget what does a gene include when i'm talking about a gene it includes the dna sequences that code for all the exons and the introns the sequences at the beginning and the end of the rna which are not translated into protein so when you're transcribing the gene you're you're gonna transcribe information upstream of the gene uh or of the exons you're gonna transcribe information downstream of the in the exons you're gonna transcribe the exons the introns so basically you're going to end up with what's called the transcription unit that means all of the mrna you're going to have information at the beginning of the mrna that you're not going to need for for a translation that's called the five prime untranslated region you're going to have a stretch of rna at the end at the three prime end uh that you're not going to need for translation that's called the three prime untranslated region that means utr untranslated region you got the five prime utr you've got the three prime utr these are untranslated regions this extra uh copying you did that's not going to lend itself to the protein and then remember in the middle you also have the exon and our and intron information as well you're not going to need the intron information you're going to splice out the intron information as well so you've got a lot of information in the transcript that you're not actually going to need for translation so you you know the gene itself includes the promoter but do you transcribe the promoter no you don't transcribe the promoter you transcribe the rna coding sequence and part of the terminator now with mature mrna you have the five prime untranslated region or utr the three prime untranslated region or utr you also have the protein coding region you have introns exons and you have what's known as the shine dalgarno sequence which is required in prokaryotes you see here the mrna of prokaryotes you have the five prime untranslated region this means stuff you copied from the gene that doesn't actually include information for making a protein and then at the three prime end of the rna you have the three prime untranslated region again this means stuff you copied from the gene that doesn't actually code for protein three prime untranslated region and then here is your coding part again this is a prokaryotic mrna so it's not going to include exons and intron information here in the coding part of the of the uh mrna here you have what's known as the shine dalgarno sequence in prokaryotes this is a sequence of nucleotides a genetic sequence that helps with initiation of translation so the ribosome to bind correctly and the ribosome to start translation here's what's known as the start codon remember a t g or a u g on the mrna would say a u g this is where translation will start translation will start and here's where translation will end remember the stop codon one of the three stop codons will live here uaa uga uag one of those three stop codons will be here a star codon will be here this is the only part of the mrna which is going to become translated into protein again downstream you have untranslated three prime untranslated five prime and what's the shine dalgarno sequence that's to help the ribosome know where to attach so you can start translation and translation occurs five prime to three prime so the ribosome would attach here it would then copy this way to produce the protein and by the way uh i have two videos for you i have a refresher video from biology 1406 um actually it's two videos in one i'm going to throw a card above right now this is a great review of transcription and translation from my biology 1406 class if you don't recall how transcription works if you don't recall how translation works these are great videos to watch i throw it up above in a card if any of this isn't making sense remember it's a review of 1406 and i have a nice video watch that video and it'll bring you back to speed so don't forget you need to in eukaryotes now this was in prokaryotes remember prokaryotes there is no rna modification that needs to occur what does that mean look at this in prokaryotes you don't need to add a five prime cap you don't need to add a three prime poly a tail you don't need to splice out introns there are no introns remember in prokaryotes transcription and translation can occur pretty much at the same time that means as transcription hasn't even completed yet translation can begin does that make sense but in eukaryotes that's not the case after transcription you have a really long piece of rna called the pre-mrna and that pre-mrna is not ready for translation it's you know in the nucleus and it's not ready for translation remember first of all it has a bunch of intron information inside which you don't need the intron information you need to splice out the intron information secondly it's highly fragile it's rna is a fragile molecule it's easily degraded so it needs to be stabilized by addition of a five prime cap and a three prime poly a tail so i'm going to explain to you what these are in a little bit but uh the addition of the five prime cap happens obviously at the five prime end of the pre-mrna and it has two functions one is to help the ribosome to know where to attach to the mature mrna and two is to stabilize and help prevent degradation of the mrna secondly a poly a tail same thing it helps to prevent the degradation of the mrna by adding you're adding 50 to 250 adenine nucleotides so like a giant tail made of a's to the three prime end of the mrna so that you prevent the easy degradation of the mrna what does the five prime cap look like the five prime cap is essentially guanine you know g the the nucleotide g guanine but it's called methyl guanine because it has a little methyl group ch3 this is methyl guanine and essentially what the five prime cap is during rna processing is taking g that has a methyl group and sticking it onto the mrna that's it you just stick g onto the mrna but there's one trick there's one catch it's a really weird capping because you're taking the five prime end of g methyl guanine the five prime phosphate and you're sticking it onto the five prime phosphate on this stretch of rna did you guys follow me the pre-mrna has a five prime end with a phosphate group and you're taking a methyl guanine and you're sticking its phosphate group onto the mrna phosphate group so it's a five prime five prime bond uh so essentially this guanine is stuck onto your pre-mrna pointing the wrong way around well that stabilizes the mrna and it helps to you know facilitate initiation of translation the ribosome to know where to start translation isn't that fascinating so that's what they mean by a five prime cap you take methyl guanine you attach it five prime five prime to the pre-mrna's five prime end and again why do you do that prevent degradation of the mrna premature degradation of the mrna and to assist with initiation of translation then you have what's known as rna splicing remember in eukaryotes you're going to have to cut the intron information out of the coding region you know the middle region of the mrna so that you're left with only the exons correct and the way your the way your cells know where to splice is these consensus sequences which indicate splice sites and these consensus sequences each splice site each intron each intron has a five prime consensus sequence of uh g u then either an a or a g a g u so it's g u either an a or a g a g u at the five prime splice site and then a three plot three prime consensus sequence of cagg and then a branch point of which consists of a right this is about 18 to 40 nucleotides upstream of this three prime this three prime splice site and then this enzyme called the spliceosome which is essentially a ribosome the spliceosome which consists of five rna molecules and about 300 different proteins binds in order to do the process of splicing so let me show you this process all right here is the process here you can see this would be part of your transcript and here between you know this light green area is your first intron you want to remove this intron remember at the five prime end of the intron you have the first consensus sequence the five prime consensus sequence at the three prime end of the c of the intron you have the other consensus sequence and then you have your branch point here about 18 to 40 nucleotides away from the three prime consensus sequence so this is where splicing of the pre-mrna occurs this is how it occurs and remember the enzyme that's responsible for doing the splicing out of the intron from the pre-mrna is the spliceosome protein concept five check if a splice site were mutated so the splicing did not take place what would be the effect on the protein by the mrna well the protein would be way longer wouldn't it because you wouldn't splice out that information and not only would the protein be longer it may not work at all because now you've got a bunch of amino acids in the protein a stretch of amino acids in the protein that don't really belong there so that most likely will destroy the function of that protein but again here's how it works your splice site at five prime splice site here your three prime splice site at the other end uh let's go through this one by one the mrna is cut at the five prime splice site so here you cut it cut then the five prime end of the intron attaches to the branch point see what happened you cut the mrna at exon one you then flopped it back and you attached it to that a you know the branch point then you cut the three prime splice site you've cut the three prime splice site and you've removed this loop you see you've removed the you've looped out the intron you've looped out the intron and this is called the lariat and true texans will know what a larry it means it's like a lasso and that's how this structure got its name it looks like a little lasso you know so the lariat is then removed the intron has been removed as a lariat as a lasso looking thing and then it's broken down and degraded the intron is broken down back into nucleotides and now you you attach the exon one to exon two so the spliceosome attaches exon one to exon two and now you're ready for translation the the spliced mrna is exported into the cytoplasm for translation you see what happened there one thing i skipped over was this slide here which is also showing you the third modification that occurs to the pre-mrna member pre-mrna and eukaryotes is modified three different ways the five prime cap which is that methyl guanine five prime five prime addition to the mrna removal of the introns with the spliceosome at the consensus sequences and then at the three prime end remember there's addition of a poly a tail 50 to 250 uh a's adenosines so let's take a quick look or um adenines instead of adding adenines so let's take a quick look at how this works so here you have your pre-mrna and where you have this consensus sequence at the three prime near the three prime end of your pre-mrna you will find this consensus sequence aaua now what happens is downstream of this consensus sequence you're going to cleave your mrna pre-mrna is cleaved at a position from 11 to 30 nucleotides downstream of this consensus sequence so you cut the pre-mrna off here then an enzyme called polyadenylase adds a bunch of uh adenines a a a poly a tail a poly a tail here and you're adding many many many many many many adenines and this tail remember is about 50 250 nucleotides long right a bunch of a's and why do you do that again it's to prevent the degradation of the uh mrna to help stabilize the mrna since since rna is a highly reactive molecule because i don't think i mentioned this before but the fact that you have those hydroxyl groups on the two prime carbon of the ribose sugar those hydroxyl groups are highly reactive which makes rna easily degraded if you're ever working in a research lab and you have an rna project where you have rna you're dealing with you'll notice that you have to take a lot of precautions because rna is so easily degraded in nature dna is much more stable and so dna is less easily degraded as rna rna is highly easily degraded so to help prevent that and to help counteract that we're adding a bunch of a's and we're adding a five prime cap all right so here's another concept you have alternative splicing in eukaryotes because you have different exons you can have a process known as alternative splicing in eukaryotes it's fascinating you have exon 1 exon 2 exon 3 after transcription what do you do you've got your pre-mrna which needs a five prime cap and a three prime poly a tail so here's your five prime cap here's your three prime poly a tail at this point what do you normally do you you loop out the introns right you you do splicing now there's different ways that you can splice in eukaryotes you could splice so that you end up with a mature mrna with exon 1 next to exon 2 next to exon 3 which is what you would expect and that's going to give you a certain protein isn't it when you cut when you translate exon 1 2 and 3 you're going to get a certain protein however one way to diversify your subset of genomic information is to shuffle exons around or let's say you could do an alternative form of splicing where let's say you splice out exon 2 as well you just you you just leave out the information for exon 2. so you form a transcript with exon 1 next to exon 3. well if you translate this you're going to end up with a different protein than if you translate this so the human genome has roughly 25 000 different genes but you have up to 80 000 plus different kinds of protein that shouldn't add up in your brain should it because remember what i said earlier a gene is the information for a protein right a gene is the information for a protein so if i were to tell you there are 25 000 different genes you would assume that there should be 25 000 different proteins because a gene codes for a protein well if how is that even possible that there's 80 000 different proteins if there's only 25 000 different genes well alternative splicing is the answer if i take the same information and i include exon 1 exon 2 and exon 3 in my transcript for for a translation i'm going to end up with a protein that includes the information for exon 1 exon 2 and exon 3. i'm going to get the amino acids for exon 1 next to the amino acids for exon 2 next to the amino acids for exon 3 information right but if i alternatively splice this this pre-mrna to exon one next to exon three i'm gonna end up with a smaller protein that only includes the information uh from exon one and exon three when i'm making my polypeptide when i make my protein does that make sense and that is a different protein so i'm essentially getting more bang for my buck i'm getting two different proteins with the same genetic information the way i i explain it to students you know in a very very simple way a simplified silly way is can't you make two different types of cookies with a chocolate chip cookie recipe um think about it i could give you a chocolate chip cookie recipe and you could make two different kinds of cookies from that chocolate chip cookie recipe couldn't you you could follow the recipe to the tea follow the recipe and what do you end up with if you include all of the information in that recipe what do you end up with you would end up with chocolate chip cookies right but what if i said okay now leave out the chocolate chip part just don't add the chocolate chip right now you just end up with nice cookies but they're not chocolate chip cookies they're just cookies does that make sense so you you just essentially made two different kinds of cookies but with the exact same information it's just you left out the the chocolate chip part in one batch and you at you included the chocolate chip part in another batch same thing here you can make different kinds of proteins simply by leaving out an exon and that exon might be a a functional group or some kind of uh uh portion of the protein that maybe a binding site on that protein or something like that you you can end up with more genetic uh variability or not genetic variability but protein variability from the same genetic information isn't that interesting prokaryotes are not capable of doing this obviously because prokaryotes don't have exons and introns so again to summarize there's a lot going on here in eukaryotes isn't there you've got your promoter uh which includes the the the binding sites at minus 10 to minus 35 you've got your exons that you need to trans transcribe so you transcribe all this information including the exons and the introns you then need to stabilize this pre-mrna with the addition of a five prime cap you then cleave the three prime end and add a poly a tail you then have to undergo rna splicing to remove all the introns as lariats and you could even splice this a different way so you get different combos the chocolate chip cookie or the regular cookie and now you're ready with your mature mrna this is called mature mrna with the five prime cap the methyl guanine cap the three prime poly a tail the introns spliced out ready to shuttle to the cytoplasm meet up with the ribosomes for translation one thing i left out was uh the promoter could also have other important sequences upstream here or way downstream here these are called enhancers enhancers are typically upstream but could be downstream and enhancers are binding sites for different proteins called transcription factors which could help transcription begin so anyway in that interesting there's what's known as pre-mrna processing five prime cap three prime poly a tail intron splicing with the spliceosome you could diversify the number of proteins you can get with alternative splicing and all of this is eukaryotic specific isn't that interesting stuff so let's talk a little bit about other types of rna not just mrna but let's talk about trna for instance the structure and processing of trna let's talk a little bit about trna and remember what is the function of trna trna plays a major role in translation it allows translation to proceed so what does trna look like trna has a couple of really rare rna nucleotide bases on it ribothyne ribothymine and pseudo-urine ureadine these are rare nucleotide bases inside of trna you don't really need to know this in too much detail but it's included here in the book and the trna itself forms a secondary structure called the clover leaf structure and that clover leaf structure includes an anticodon where the binding occurs between mrna and trna so here you can see this is a trna and you can see the same trna in three different forms here you see each individual nucleotide of the trna and what's binding to what remember that trna has a or like our all rna has a five prime end and a three prime end but its secondary structure can form hair pins like this and it's called the clover leaf because this kind of has a clover leaf structure now when you look at the ribbon model you can see how it folds and when you look at the space filling model you can see its overall shape so this is called the space filling model where you can see each individual uh each individual atom here this is the ribbon model showing you the backbone of the dn of the rna in this case the rna and here you can see this is the flattened model this is just showing you the individual nucleotides of the trna now let's talk a little bit about some of the features of trna you have the rare bases i told you about the rare bases you have the anticodon the anticodon when they talk about the anticodon loop they're talking about these three nucleotides one two three this is called the anticodon it will bind to the codons so do you remember the start codon for instance a u g right a u g on the mrna the anticodon on a trna would read u a u so u a c u a c the anticodon for uac would bind to the codon aug and that would initiate translation for instance so the anticodons on the trna bind to the codons on the mrna in order to facilitate translation the other important part of the trna is this three prime end this is called the amino acid attachment site which is always cca this is where the trna is charged with an amino acid and we're going to talk about this in the next chapter we're going to talk about translation but here's where the amino acid is attached to the trna at the three prime end cca here's where the trna binds to the mrna in a complementary fashion with its anticodon loop okay so those are the two important things to know about trna next we're going to talk about rrna rrna ribosomal rna did you know the ribosome which facilitates uh translation the ribosome is part protein and part rna the rna part of the ribosome is called rrna for ribosomal rna so let's talk about that remember there's two subunits to the ribosome the large ribosomal subunit and the small ribosomal subunit in bacteria the large subunit is called the 50s subunit and the small subunit is called the 30s subunit they come together i i give the analogy like a hamburger bun right the large hamburger bun attaches to this small hamburger bun to form the hamburger buns together that is the 70s ribosome so in bacteria it's called the 70s ribosome and in eukaryote it's the ads which the large subunit is called the 60s subunit the small subunits called the 40s subunit they come together to form the ads subunit by the way what did i tell you i told you that the ribosomes are part rna and part dna so look at this the large subunit in bacteria is made up of uh what's known as the 23 s of rrna this is a a long piece of rna and then the 5s rrna here's a shorter piece of rna so two pieces of rna two pieces of rna make up the large subunit of the bacterial ribosome as well as 31 proteins did you catch that 31 proteins plus two pieces of rna equals the large subunit of the bacterial ribosome 21 proteins and and one long piece of rna called the 16s rna make up the small subunit of the ribosome in eukaryotes 49 proteins along with one two three three pieces of of rrna make up the large subunit and 33 proteins along with a piece of rna make up the small so again the large subunit and the small subunit of ribosome is a ribosome it includes protein and rna let's talk about another type of rna actually two different types micro rnas and small interfering or si rnas mi rnas and si rnas micro rnas are amazingly interesting these are part of your own genome basically they look like genes but after transcription these micro rnas pair with themselves so instead of coding for a protein these micro rnas will kind of form a hair pin and then this pre micro rna is cleaved to produce a short rna with a hair pin so you cleave the hair pin off this is your micro rna and that could lead to a process known as gene silencing you can then use this micro rna to turn off expression of genes same thing with small interfering rnas you have double strand rna the double strand rna is cleaved by an enzyme called dicer into small rnas or small interfering rnas these are small double strand rnas and that could lead to gene silencing so look here the micro micro rnas cut with dicer one strand of the microrna combines with proteins to form an rna inducing silencing complex called risk risk which is basically a protein plus that you know micro rna and then there's imperfect base pairing between the micro rna and the mrna message of a gene causes inhibition of translation so this gums up the works micro rnas prevent translation of complementary mrna transcripts so it's a way of turning off gene expression post-transcriptional inhibition of gene transcription isn't that interesting small interfering rnas are the same uh but the outcomes a little different with small interfering rnas the si rna combines with the risk so and in this case there's perfect pairing and that leads to cleavage and degradation of the transcript so either way micrornas and small interfering rnas either way they stop uh transcript they stop translation they stop gene expression so you can turn off the expression of a gene using micro rnas or small interfering rnas uh and and so it's an interesting process sometimes this is done naturally there are micro rnas in your body right now like in your heart that that need to be transcribed in order to regulate proper heart development and function and then these can also be used in biotechnology to knock down expression or reduce expression of different proteins to see what happens in the in the lab as well so it's really fascinating stuff this whole world of small uh interfering rnas and micro rnas started with andrew fire maybe about 15 years ago it's it's relatively new area of study but very fascinating stuff we're still learning the benefits of micro rnas and small interfering rnas today we're still trying to figure out exactly why they're so important again here's the differences between s i rnas mirnas you go over this and lastly on this chapter i'm going to tell you about the the newest type of rna which we've learned about which is fascinating over the last 10 years we've started to learn about crispr rnas you may have heard of crispr in the news about how crispr technology is again lead us towards these genetically modified organ organisms and genomes and how it's going to fix all the problems or you know lead to uh designer babies and such we're going to learn about this we're going to learn a lot about this but it's a it's essentially a rudimentary immune system in prokaryotes isn't that fascinating we're going to talk about this a lot but um basically what a prokaryote can do is take some foreign dna some foreign dna like phage dna fade like bacteriophage dna it could acquire this phage dna as a spacer it can place it as a split spacer in between these palindromic sequences and then that becomes part of its repertoire uh its immune system if you will and and what crispr rna does is that it will transcribe these uh these spacers this space essentially sequences from infections past and those transcripts will bind to the cass protein which is part of the crispr system forming an effector complex the effector complexes the cast protein plus this this uh this spacer this spacer um information from a phage from past and then if the foreign dna if that phage were to infect the bacteria again the cast protein would be directed towards that dna because of binding between the foreign dna and the spacer dna that remembers that binds to that binds to the foreign dna so if that phage were to infect that bacteria again that bacteria would be able to attach to the phage dna and cause cleavage and destroy that phage dna isn't that interesting so it's a rudimentary immune system in bacteria when when a bacteria becomes infected it has the opportunity to incorporate some of that infected um some of that infectious dna into its own dna as a spacer unit you know and and all of these spacers represent information from infections past those are transcribed paired with casts and casts can then attack and degrade uh that foreign dna if it were ever to infect the cell again isn't that fascinating we always thought up until like when when we discovered crispr we always thought bacteria are so rudimentary they they don't have any immune system but this is their rudimentary immune system isn't that fascinating stuff just like we never thought they had sex right that bacteria can't do sexual reproduction but then we learned about conjugation and the sex pilus you know and just like we thought bacteria couldn't communicate because how would the bacteria that can't see hear taste smell how can this bacteria communicate but then we learned about quorum sensing and they're fascinating stuff that you under you underestimate life and then it shows you uh the difference there anyway fascinating chapter last last slide before we call it quits this is our uh you know our uh stats sheet our epic stats sheet for this model organism the nematode worm cano habitus uh elegance and many many labs studied this worm it's a transparent worm so you can see right through it they know exactly how many cells are in this worm they know exactly how how many uh neurons are in this worm and what each neuron does so it's a wonderful worm to study it's a wonderful wonderful model organism for study so it it's not it's wonderful because it's small size it's easy to grow in the lab as a very short generation time you can carry 200 to 1 000 eggs per female or each female can produce 200 to 1 000 eggs so a lot of proliferation going on there easy to culture simple body plan transparent capable of self fertilization so they're hermaphroditic round worms they're very small you can grow them on plates they have chromosomes you could study and why do we study them genetics of development apoptosis program cell death genetic control of behavior aging a lot of neuronal studies neurons and you want to know something fascinating about the nematode that has to do with s i rna i'm going to tell you something fascinating remember i told you let me tell you a little story look at the si rna what do they do double strand dna that pair up with the the um proteins to form the risk complex and the risk complex shuts off uh you know translation or expression of a gene right you know what they do in research labs with these worms they take s i rnas they put them in e coli right in bacteria they then feed those e coli to these worms these worms eat the e coli that have the s-i-r-n-a and then that s-i-r-n-a knocks down expression of genes in the worm isn't that fascinating so if you want to study how a particular neuron works in this worm you could make an sirna that's targeted against one of its neural genes put that sirna plasmid inside of an e coli have the worm eat the e coli and then the worm will have not only ate the e coli but ingested the s i rna against its neural cells it will knock down neural expression of that neural gene target and then you could study what are the repercussions of knocking down that gene in the worm and that fascinating stuff i mean tell me that's not fascinating anyway that leads us to the end of this chapter i hope it was interesting it's very uh interesting stuff but i could tell that you know it is it is getting kind of dense with material molecular biology material hopefully it made sense though let me know if you have any questions in the comment box but below and as always i will catch you guys next time dr d dr d dr d dr d dr d dr d a dr d dr d dr dr d dr d dr dr d dr d dr dr [Music] dr d