hi welcome back to video three in this third video we're going to look at differences between procaryotic and eukariotic transcription as well as the types of processing needed in ukar before messenger RNA is formed so transcription in procaryotes and UK carots is very similar but the main difference is that because in ukar we have a membranebound nucleus this means that transcription will occur in the nucleus where we read our DNA and we make our messenger RNA then it has to be transported the messenger RNA has to be transported to the cytoplasm where ribosomes will bind and translate the messenger RNA into protein because of this process and transportation of the messenger RNA we also have to make sure that messenger RNA is protected and not degraded before it's translated to do this we actually produced premrna first and nucleus and there's going to be a few steps required known as mRNA processing before we form our final messenger RNA molecule that's transported to the cytoplasm we're going to see that ukar also use three different types of RNA polymerases that transcribe different types of genes and finally in ukar the RNA the messenger RNA is usually monogenic which means that a single messenger RNA molecule usually encodes just a single protein in contrast we already saw this earlier but in procaryotes like bacteria there's no nucleus so transcription and translation can occur simultaneously their messenger RNA is degraded much more rapidly because it's not protected like we're going to see soon for UK carots it is protected and in procaryotes their messenger RNA is usually polygenic where you'll see on a single messenger RNA molecule if this is a single mRNA you might see several genes like Gene one gene 2 and Gene 3 all on a single messenger RNA molecule eukariotic transcription is much more complex and in fact in ukar we have three different types of polymerases depending on the kind of Gene you're going to be transcribing RNA polymerase will be transcribing ribosomal RNA genes RNA polymerase number two will be transcribing our protein coding genes and this is the one we're going to mainly be focusing on for transcription and finally RNA polymerase 3 transcribes ribosomal RNA Transfer RNA and small nuclear RNA genes and in addition to RNA polymerase it's not shown in the slide but each type of RNA polymerase and you Nuuk carots also requires transcription factors transcription factors in order to bind to the DNA template strand sometimes we abbreviate these TF for transcription factors if we look at the first step of transcription or initiation in ukar the promoter in UK carots is much more uh it's much larger and more complex than procaryotic Motors that we saw earlier but there is a sequence that we saw earlier in procaryotes it was around minus10 nucleotides Upstream from the initiation start site in UK carots it's located around Theus 25 to-35 sequence or uh bases Upstream of the initiation site and that's known as the Tata box this is our consensus sequence and UK carots and the sequence is TA a TA AA we see the sequence in pretty much all UK carotic cells and that's always Lo located within the promoter this sequence is what our transcription Factor transcription Factor recognizes and binds to and this is the first transcription Factor that's laid down the transcription Factor you can see includes a subunit over here called tbp and that stands for Tata binding protein binding protein you don't have to know the small numbers or letters here but know that once those come down and bind to the tatab Box more transcription factors are recruited until ultimately RNA polymerase 2 is recruited and binds to the promoter near the region of the TT box so here the entire complex with those transcription factors and RNA polymerase 2 forms our transcription initiation complex and remember RNA polymerase 2 is the type of polymerase we had three different types this is the type that transcribes protein coding genes on the DNA so right under this image in our Open Stacks textbook there's a question where they ask a scientist splices a UK cartic promoter this promoter in front of a bacterial Gene so let's pretend there is a bacterial Gene around here and inserts this whole thing the promoter and the bacterial gene into a bacterial chromosome the question is would you expect the bacteria to transcribe the gene so if I had a eukariotic promoter in front of a procaryotic or bacterial Gene would the DNA be read and form messenger RNA would I be able to transcribe the Gene and the answer would be no it would not work because UK carotic promoters will not be recognized by RNA polymerase of procaryotes procaryotes use different promoters than ukar so this will not work if you try to mix a eukariotic promoter with a procaryotic gene for a second step of transcription elongation there are also differences compared to procaryotes in ukar it's similar to procaryotes in that messenger RNA is synthesized in the direction of 5 to 3 Prime but this is going to happen in the nucleus of ukar whereas in procaryotes it's happening in the cytoplasm since they don't have a nucleus additionally because you carots because we wrap our DNA around histone proteins and it's pretty tightly bound and inaccessible we actually have to make sure we loosen the DNA move it kind of away from the histone so that it can can be accessed by RNA polymerase during transcription and then we have to rewind it retighten it around the histone proteins after transcription is over and this is facilitated by fact proteins a fact complex and that stands for facilitates chromatin transcription that's the protein complex name so it basically moves along the chromatin and loosens again unwraps the DNA loosens it so it moves away from the histone proteins and then as you're done with a certain region it retiens it reforms that nucleosome complex finally the last step of transcription our termination step is different in UK carots because for AR polymerase 2 which transcribes our protein coding genes we don't have what we saw in procaryotes there's no row dependent or row independent termination instead RNA plase 2 is actually going to transcribe thousands of nucleotides after the gene template so it just keeps transcribing and ultimately these nucleotides are going to be removed when we're processing our messenger RNA when we're turning it from premessenger RNA into our final messenger RNA molecule if I look at RNA polymerase 1 there are also differences and termination RNA polymerase 1 will recognize or require excuse me a termination Protein that's going to recognize a specific sequence to stop transcription and finally Arna polymerase 3 is going to form a hair pin complex that's similar to what we saw in procaryotes we're going to focus mainly on RNA polymerase 2 again for our class one of the key differences between procaryotes and ukar is that in procaryotes when you make your messenger RNA it's ready to go and be translated right away in UK carots however the premessenger RNA that's formed during transcription is not ready to be translated yet instead it has to go through processing before you form your messenger RNA that's ready for translation so let's look at the three things that have to be done during this processing the first step is something known as intron splicing we're going to have to cut out or remove sequences known as introns these are intervening non-coding sequences that we get that are going to get cut out the second thing we're going to do is add a five Prime methyl guanosine cap and that's sometimes called a g a gcap we'll see that at the five Prime end of the messenger AR RNA so that's usually depicted on the left hand side of our messenger RNA molecule the final thing we're going to do is add a three prime polyat tail on the three prime end again of the messenger RNA so you'll see a bunch of addine nucleotides having the five Prime gcap and that thre Prime polyat tale actually increases the halflife of the eukariotic MRNA so it doesn't get degraded as quick L as it does in procaryotes and in fact while in UK carots the messenger RNA can last for hours so translation can keep occurring in procaryotes like eoli the messenger RNA only lasts for a few seconds like maybe 5 seconds or less so here's another look at that RNA processing we start with our premessenger RNA and remember in ukots we have to go through extensive processing the first thing we're going to do is cut out or remove the introns so the introns are intervening sequences that are not coding they're non-coding so those are going to be cut out and removed we're going to keep regions called the exons so exons are called exons because they are expressed these are the important regions of messenger RNA then again we're going to add our five Prime gcap and our poly a tail on the three prime end and that's going to allow the messenger RNA to be stable and not be degraded by different enzymes found within the cell so here is my final messenger RNA molecule that's ready for translation you can see the exons have been retained the introns have been removed and I can see that five Prime end at 3 Prime end that have been protected so enzymes don't degrade the messenger RNA molecule notice how small the final messenger RNA product is now that you've cut out all of those introns those intervening sequences so here's another look at the same thing except we're going to focus on the vocab terms we have exons which are protein coding sequences that are expressed and we're going to keep those in our messenger RNA molecule so this is really our pre mRNA and then what we're going to get rid of are the introns the introns are the intervening sequences that must be removed we do this through a process known as splicing so splicing will allow us to remove introns and reconnect the exons together notice that we have our five Prime gcap over here and our poly a tail over here and we're not going to look at this yet but in a later chapter we'll see that there are untranslated regions on both both ends of the MRNA as well there's a five Prime untranslated region and a thre Prime untranslated region it'll be important in our next chapter so splicing is that removal of those introns from our premrna transcript and it's catalyzed by these large protein complexes known as spliceosomes spliceosomes over here these spliceosomes are made up of proteins and in combination with small nuclear RNA molecules so SN rnas and this one should there's a typo there so this should say SN rnas and I can see that the spliceosome which is this whole complex is going to bind to and get rid of my intron which is in green right here although the intron is non-coding it's an inating non-coating sequence there are marks at the ends of the introns so the five Prime end of the intron has a g and U nucleotides and the three prime end of these introns have a and g at their end and spliceosomes will recognize these two sequences bind to that region to remove the intron over there here's another look at the same thing in this image the whole thing over here is the spos and we can see these small molecules are those small nuclear rnas this silver segment right here is the intron that we're going to splice out and these would be over here would be the five Prime and three prime end of the introns that are recognized by the spos if we have mutations in the DNA which will lead to mutations in the messenger RNA that could lead to splicing errors so if these sequences were lost these recognition sequences for the spliceosome were lost then we might not be able to cut out the intron and that could lead to a protein that ultimately becomes nonfunctional or if there are mutations in the small nuclear RNA sequences that are part of the spliceosome that could also cause problems with cutting out introns and producing normal proteins after you translate the the messenger RNA just as you did for the previous two videos after you finished watching this third video on our canvas website you'll see an external link that'll take you to this animation page I'd like you to click through the animation it's very short it reviews the process of RNA splicing and the processing of premna so our premessenger r RNA will be processed converted to messenger RNA and then translated but ribosomal rnas and transfer rnas are not going to be translated they stay in their RNA state pre-ribosomal rnas will be transcribed and processed and they're going to be assembled into the ribosomes within the nucleolus remember the structure is found within the nucleus pre-transfer rnas are going to be transcribed and processed in the nucleus then they're going to be shuttled to the cytoplasm and they're going to be bound to free amino acids to help with protein synthesis so this is a picture from our textbook that shows us the image or the structure of Transfer RNA and the function of Transfer RNA is to bring amino acids to the growing polypeptide chain during translation so we'll see these in the next video they're going to be interacting with ribosomes to help grow our polypeptide chain for the TRNA there are two important sites one is the amino acid attachment site and in this case it happens to be the amino acid pheny alanine on the other side we have an anti-codon that will base pair or hydrogen bond with the codon that are found or the codons that are found on the messenger RNA so if this is the anti-codon then the codon would be u u and this is what encodes feny alanine so if we go back and look at that codeon chart the genetic code you'll see that uuc on the messenger RNA encodes fenal alanine for its amino acid and this is from a different textbook but I wanted to show you other images or depictions of Transfer RNA sometimes you'll see something that looks more like this this is where the amino acid would be bound and over here is where the anti-codon is located or sometimes you'll see something like this again this is where amino acid would be bound and here's my anti-codon if the anti-codon is ACC what do you think the amino acid is so I would give you the code on chart let's see in the next slide so here is my codeon chart again this is the genetic code if this is my anti-codon then what that means is I know my messenger RNA where the codons are housed would be complementary so that would be a pairs with u that would be G G and if I look at my codon chart u g g u g g encodes the amino acid tryptophan so that's the amino acid that would be attached on the other side of the transfer RNA molecule so notice that the amino acid is determined by the codon not the anti-codon so we look at the codon chart here right and that takes us to the end of our third video in our fourth and final video for chapter 15 we're going to be looking at the details of translation how do we go from messenger RNA to our final polypeptide or protein product