TRANSCRIPTION

this session or last week we've learned the process of DNA replication we learned that DNA replication is a process by which um parent cells transmit their genetic information to daughter cells in other words there's a mechanism of heredity transmission of uh genetic information from generation to generation that exactly is why say for example you have characteristics or you share characteristics with your parents you might you might have your father's eyes or your mother's nose and perhaps um you also have some inherited characteristics like you have and hopefully not you have uh fair I mean you have this hopefully not as I'm saying hopefully not you have some metabolic disorders that are um that are associated or known in one in one in in your maternal or paternal branch of the family or side of the family for example but again hopefully not so the the the the process of replication is just one of the one of the three steps um that comprise the central dogma of molecular genetics or molecular biology now we're going to study the process of transcription and uh see for ourselves how this genetic information is a transcribed into a working copy that is then ultimately translated into proteins remember what you have in your genome is your genotype how it is expressed and how it therefore manifests in individual is determined by its phenotype you have to remember that not all of the genetic information that the celf processus is expressed and ultimately translated into proteins much of the genome is actually junk DNA it is not expressed is not translated So today we're going to study the process of transcription it is the uh process by which the genetic information in genome is transcribed into messenger RNA or into mRNA transcription of a genetic code all right now um here are the general features of RNA synthesis the important features of RNA synthesis and you will notice in the course of our discussion that many of this features especially the action of the polymerases are are similar to that of the DNA polymerase excuse me so the RNA is initially synthesized using DNA as a template in a process called transcription enzyme that catalyzes the process it's DNA dependent RNA polymerase it is DNA dependent RNA polymerase because it uses DNA as your um subst all right um similar to the case of DNA replication there has to be four ribonucleoside triphosphate precursors or the nucleotide precursors in this case instead of having thyine as the in one of the nucleotides you have uril as one of the nucle as the base in one of the nucleotides so you have ATP GTP CTP and UTP remember in DNA a pairs with t and t pairs with a in RNA synthesis a does not pair with t instead a pairs with u okay but T still pairs with a so that means means that the RNA transcript does not have any thyine residue okay a primer is not deleted in RNA synesis remember that in the case of DNA synesis it has to be an RNA primer it's introduced by the primas remember primas is a specialized RNA polymerase um that introduces RNA to the um um the growing either the leading or the lagging strand um I mean has to be put first into the leading or the lagging strand in order to synthesize a complimentary DNA strand in the case of RNA polymerase that is not necessary because the RNA polymerase can initiate and elongate the developing RNA primer is not needed in the RNA synthesis but a DNA template is required as in the case of DNA biosynthesis process is elongating or rather the process elongates a chain from the five Prime to three prime direction right from the five Prime to three prime Direction the nucleotide at the five Prime end of a chain retains its triphosphate group we abbreviate that as PPP meaning triphosphate they're three phosphorus or phosphate groups the enzyme uses one strand of the DNA as a template for RNA synthesis and here it is you have to um be aware and I think you already realized that by now the DNA consist of a duplex remember it has you it has two strands the question is which of these strands is going to be um transcribed right into which of this is going to be um transcribed into a working copy in other words which of this two strands is going to be copied into an mRNA transcript well the answer is both of them right but the information contained in one strand is not the same as the information contained in the other strand all right some of um information can be found in one strand the other can be found in a complimentary strand okay in fact that segment of the or that segment of a strand yes that sequence or segment of the Strand that codes for a particular protein or for a set of proteins is called a gene I believe you've encountered the term Gene before right the Gene g n g NE e it's um a sequence or a segment of a DNA strand that codes for a protein or for a family not a family actually for for a set of proteins that are important for say a metabolic uh process like a metabolic reaction um so these two strands can be transcribed and we're going to clarify this more more in this later going to discuss more in this later but I want you to remember that of the strands that are being trans The Strand that is being transcribed is called a template strand it's also called the negative strand it's also called The anti-in Strand okay um so as uh mentioned here the base sequence of the DNA contains signals for initiation and termination of RNA syesis enzyme binds of a template strand it moves along it in the three prime to five Prime Direction the template is unchanged okay now don't worry we're going to uh discuss each of them each of those processes in detail later as uh before um the process of transcription is of course um best understood in procot um like the eoli so we're going to focus our attention on our on on transcription in in procaryotes eoline this okay so molecular weight here is about the RNA polymerase talking about the RNA polymerase the enzyme that in synthesizes RNA so the molecular weight is about 500 kilodaltons okay there are four different types of subunits the alpha beta beta Prime and sigma all right now I want you I want to emphasize that it's the alpha the beta and the beta Prime that comprises the core enzyme the core enzyme is the enzyme that actually does the catalysis it actually um catalyzes the synthesis of the RNA the sigma as you can see here Sigma is actually that part or that uh subunit that um that binds to the core enzyme and uh and and to form the Holo enzyme now the the sigma is the uh subunit that um bind that recognizes the um sequence in the are in the DNA that needs or that is to be transcribed all right the uh Sigma it's the one that recognizes the uh promoter region the promoter region is a region that signals the RNA polymerase to to uh transcribe that particular Gene okay all right now let us say let us discuss more about the promoter the promo there is a long DNA I mean your your DNA strand is long right how do you know which of these which part or which segment of this um TNA strand is going to be transcribed right that question is answered by the promoters promoters are sequences in the DNA that as the name itself suggests promotes the transcription of uh of a particular segment of the uh DNA okay and the sigma the sigma subunit let me use my pointer the sigma subunit here recognizes that promoter region and therefore binds to that promoter region and uh after which the core enzyme binds to the sigma subunit and then the polymerase then starts to transcribe that particular segment of the DNA we're going to discuss more on that later the role of a sigma subunit is the recognition of the promoter Locus as I've mentioned already the promoter Legion the sigma subunit is released after the transcription begins all right okay here is what I'm talking about of two DNA strands the one that serve as a template for RNA synthesis is called the template strand or anti-sense strand all right the other is called the coding strand or the sense strand or the positive strand okay a non-template strand okay Holo enzyme binds two and transcribes only the template strand now here is where students or tend to get confused so be very careful I'm going to emphasize it now remember that the um um RNA polymerase synthesizes RNA from the five Prime to three Prim I mean extends RNA synthesizes RNA in the five Prime to three prime direction right so that means that it's template should always being the three prime to five Prime so this is an RNA transcript I hope you can see that the RNA transcript is being formed it's five Prime to 3 Prime all right five Prime to 3 Prime so it's using as its template the three prime to five Prime this one three prime to five Prime strand of the DNA right that strand is called the template strand look at my my my laser pointer this strand is called the template strand the template strand it's also called The anti-sense Strand or it's also called the negative strand now what you're going to notice is that the Strand that is the DNA strand that is comp mentary to the template strand is called the coding strand well remember the coding strand is not the template but you will remember that the that the um that the uh mRNA is actually um complimentary to the DNA the only difference is that instead of having T it has U right we call this the non The non-template Strand but the Strand of the DNA but is not um being transcribed in this gate as the coding strand because its sequence look at that five Prime to three prime is actually the same as as the sequence of the RNA transcript right the only difference is that instead of t look at that t you have U again instead of T you have U instead of T excuse me you have U U instead of T you have um U all right now that is why we would call it a coding strand because the coding strand that is the strand of a DNA that is not being transcribed is actually um actually has the same sequence as the MRNA transcript is being formed or it is being synthesized the only difference again is that instead of having a t the MRNA strand that is being synthesized has U okay all right so I hope that that is clear now the process of transcription generates is RNA transcript which is then translated see here this mRNA transcript is then translated you have protein and amino acid one amino acid 2 amino acid 3 amino acid four and so on so forth we're going to learn more about that when we study translation now let's go back to okay I hope that that is clear all right now you're going to ask me okay in this particular instance it is this trend that is serving as the template Strand and is being transcribed right and you're going to ask sir is it possible for the other strand this strand to be um um transcribe right it is possible it is possible for this non-template strand to be the template strand but that will be the template strand for another Gene right for another Gene of another Gene product and since this is in the five Prime look at that five Prime to three prime Direction your RNA polymerase must move must synthesize the um mRNA transcript from here look at my laser pointer from here to that direction perhaps I should uh use the uh pen okay so Mr I'm sorry what am I doing eraser so what I'm trying to say is that I'm just trying to draw an arrow okay that is a direction of a cesis of your RNA polymerase in the opposite direction because it uses again it extends it from five Prime to three Prime so it um template must be here from uh three prime to five Prime okay so the question is can the non-template Strand be the template strand definitely but it would have to be the template strand for another Gene for another Gene for the expression of another Gene okay all right now as I've mentioned before there exists sequences of DNA that that uh makes possible the uh expression of a particular Gene product like you're going to ask ourselves a question as we already mentioned that before how does how does the cell know which segment of the DNA is going to transcribe right it knows that by recognizing particular regions in your DNA called the promoter region or the promoter Locus this promoter region of this prmoter Locus um signifies that signifies of RNA polymerase that there is the but close to to close to it is the is this is a transcription start site or in other words it signals the RNA polymeris as say hey come here here is where the gene that you need to express here is where the gene that you need to transcribe okay okay the promoter sequence let us use my pointer the simplest organisms contain a lot of DNA but it's not transcribed as I've already mentioned this is junk DNA right um RNA polymerase needs to know which strand is a template Strand and which part to transcribe all right and where are the first nucleotide of Gene to be transcribed is okay and this is um taken care of by the promoters promoter are DNA sequences should provide direction for the RNA polymerase right okay now the the the um the promoter sequence have been determined for different genes for different genes and they found a consensus sequence when you say consensus it's it means something of a of an commonality all right there is something in common about this promoter region so this is the promoter sequence or we also call that the promoter region we have here they they measured or rather they sequence the promoter regions or different Gene like have your Gene ARA b a ara C these are just protein products all right um Lac la ale gal P2 these are just protein products and they notice that there is some common features that are present in the promoter region which is of course not surprising the RNA polymer have some variation from species to species or it even has some variations from from some minor variations from cell to cell but there has to be some kind of a common feature that is recognized by this RNA polymerase all right um so they found out that if this is your this is the promoter region now this is transcription start side TSS what does that mean it means that the trans description starts at that particular at that particular um base now here is the thing and here is where our students tend to get confused so please bear in mind and please be very careful okay I'm going listen very carefully to what I'm going to say this sequence is not the sequence of the template Str okay I'm going to repeat that this sequence is not the sequence of the template strand this is the sequence of the of the what of the coding strand H going to ask what are you talking about Sir so I've already mentioned this is the template strand remember if that's a template strand it's from three prime to five Prime now the coding strand is the Strand that is complementary to the template Strand and when we're talking about um the sequences like the uh promoter sequence we're not actually and it's it's quite funny at first and confusing actually um we're not talking about this template strand we're talking about the coding strand it is simply um out of convention right it has been agreed upon that that's what we're going to do and that it's not without reason because this um uh again coding strand has the same sequence as the MRNA trans trans with the exception of course the t's replace the U's okay I hope that that is clear again the sequence that is being shown here is or the sequences that are being shown here are not the sequence of the template strand but the sequence of the coding strand all right okay I hope that is clear and when you say Upstream that means okay let's see when you say Upstream this is that so that means it's the near five Prime all right the five Prime Direction you say Downstream is in the three prime Direction so here I think I was talking about the transcription start side before I digressed so the transcription start side here is given the number plus one plus one that's that indicates the um position of the base but is transcribed all right so in this case for example for r b a a is the transcription starts right for AR C it is G and for Bio a for Gene bio a it is a for Gene bio B it is T and for Gan do P2 it is a okay that's the transcription starts side and if that's positive one then this should be look at that positive 1 this is negative 1 -2 -34 56789 -1 all right so there's no zero in other words we don't have any zero in this notation scheme okay now they found out that there is a consensus sequence there are actually two consensus sequence one is approximately found around the -10 region and that is called the pral the prol Box the prol box right can see here why is it called consensus because look at that they have they are not the same naturally but they're almost the same t a c t GT T GTC a t this is a sequence that is consensus or that is common almost the same for all of its different genes all right there is another consensus sequence in an upstream position approximately thetive 35 region this is that consensus sequence now um and you're try going to tell me surve are not exactly the same yes but is true and they they need not to be exactly the same they just need to be similar right okay so the consensus sequence all right here the percentage occurrence of the indicated base shown here at the 35 region T all right what do you mean t42 c38 t82 what does that number means it means that you have 42% chances of occurring the th mean 38% chances it is C 82% is f mean and 84% is T that particular region right the negative 35 region in the pral box it is pro it is most probably 79% I mean here and here the a is 95% as you can see look at that it's almost a right see that it's almost a always and you have here T is 44% and so on and so forth um what is this 11 to 15 base pairs it corresponds to this one the uh um the region or sequence of the coding coding strand that um separates the Primal box one consensus sequence and the other the other consensus sequence all right look at the transcription start side okay the transcription start side the chances of the transcription start side um is a is 51% I mean is 48 um G is 42 and C is 55% so it it's it's just chances of it being that particular base so what I want to emphasize at this point in time and what I want to be clear to you is the fact that Thea first and foremost that the promoters are sequences or segments of DNA that signal the RNA polymerase signal to AR polymerize that here or close to it is the gene to be trans described all right and second that this promoter regions have consensus sequences in other words conserved sequences sequences that are almost always almost all but are almost the same um for different genes and that lastly the way to shown here is the sequence of the coding strand not the template strand all right I hope that is clear all right now how does the process of transcription transcription occur or how does it happens as I've mentioned before uh I think I haven't mentioned that I'm sorry the RNA polymerase consist is actually a hollow enzyme well I think I did Let's uh go back here it's Hollow enzyme right and it consists of two alpha subunits One beta one beta Prime and one Sigma I'm talking about the RNA polymerase in eoli ini a shakol so two alpha subunits beta and then beta Prime subunit and then Sigma subunit as I've mentioned already the sigma the sigma subunit is not necessary for catalysis it is not necessary for catalyses it's just um it's present for an entirely different purpose what is necessary for catalysis is the core enzyme Alpha 2 Beta beta Prime all right okay so how then there's a process of transcription begin process of transcription begins first by initiation all right what happens is that the initiation begins when RNA polymerase binds to promoter and forms a closed complex after this the DNA unwinds the promoter to form open complex which just required the chain elongation now here let me um um see it's too small okay so here is the initiation and elongation in the process of transcription um this one is RNA polymerase look at that you have a a brown colored seg or region and um a green colored region the green colored subunit is actually the sigma subunit that green that brown colored region is actually the um Alpha 2 Beta beta Prime the core enzy in other words this is the Holo enzy now the sigma subunit is necessary for recognizing the promoter all right necessary for recognizing the promoter okay once it binds to the promoter region then the process of transcription can begin okay now here's the thing that I want to to uh um bear in mind first and foremost as I've already mentioned much of a DNA is junk it is not expressed right it is not expressed and that genes are not expressed they're not transcribed and translated to the same extent some genes are being transcribed more often and Gene products are being synthesized at a much faster rate than other Gene products right why because your genetic your your cell is responsive to its environment it will only transcribe those genes with the cells actually need at that particular moment in time all right it will not waste energy trying to synthesize a gene product or A protein that it does not need at the moment okay and also the fact that um fact that some genes are expressed more than others suggest that their promoter region what their promoter region varies in their affinities with with RNA polymerase Sigma subunit some promoter bind the RNA polymerase Sigma subunit weekly and therefore is not transcribed as much as as as as Gene Gene whose promoters bind tightly to to uh the irona polymer a sigma subunit right so this differences in the Affinity of the uh Sigma subunit for the particular sequence particular sequence of the um promoter region accounts for for in fact that different proteins or Gene products are expressed at different or to different extents okay um all right now as I've been mention as I've been saying the recogn of promoter by Sigma as you can see here Sigma subunit then binding of polymerize Hol enzyme to DNA all right and then migration to promoter so it recognizes the promoter and binds to it now the step two we have the formation of RNA polymerase close promoter complex what do you mean close I mean it's close remember the DNA duplex is a um exist um what do you call it it's it's coiled right there's that Alpha Helix there is I mean this helical structure I'm sorry I mean I meant helical structure this uh helical structure of the DNA So when you say closed it's still um double helical all right you need to unwind it the same as in the process of replication you need to unwind it otherwise you won't won't be able to to uh synthesize mRNA so the second step is a formation of an RNA Prat close promoter complex all right the Second Step naturally would be the unwinding unwinding of DNA at promoter and formation of an open promoter complex so what happened here they just unwounded it so it has been unwounded okay then what happens next the RNA polymerase initiates Mr a synesis and it's almost always starts with a purine like adenine and guanine okay now I want you to notice something um um okay now it's almost always adenine or or guanine it's a purine now what do you notice the first um nucleotide that is incorporated always has this PPP what is that the triphosphate remember that always ask that PPP or the triphosphate and that is at the five Prime end remember five Prime okay when the RNA polymerase Hollow enzyme catalyzed elongation of mRNA by about four more nucleotides so see that I'm not sure if you can see that it's quite small but it's n nnn the one the blue box is and and and and that means it's a nucleotide and again it's extending it in the five Prime to three prime Direction three prime hydroxy group of the um ribon nucleotide attacks the five Prime phosphate I mean the the Pyro the triphosphate five Prime and all right of the another nucleotide nucleotide but is being added and what do you notice here after elongating about four more nucleotides the sigma subunit just dissociates right why remember the Sigma subunit is necessary for recognizing the promoter once it has already perform its function the enzyme or the the uh transcription no longer has need for it right it no longer has need for it so it is simply released into the nucleus the nuclear nuclear um um what do you call a nuclear space all right huh um that is very interesting because what does that mean that Sigma subunit can then be recycled and used recycled and used by what used by other um RNA polymerase core enzyme in transcribing other segments of the DNA other genes right okay and so your RNA polymerase just keeps on um elongating the RNA a transcript I hope that that is clear okay so let me ask you a question before I continue what will happen say for example if uh the um there is a problem with the sigma sub unit like uh if your Sigma sub unit is uh unable to buy to the RNA to the um to the uh promoter region okay let me all right now after the strands and this is some we've already gone through this after the strands are separated a transcription bubble of approximately 17 base bear moves down the DNA sequence to be transcribed like um here is what I'm talking about transcription bubble remember the DNA is wound and so you have here it's that transcription bubble the problem is that as in the case of a DNA replication when you unwind the DNA you're going to form positive super coils Downstream in other words ahead this is not the replication fork this is a transcription bubble you're going to form positive super coil here ahead of the uh here ahead of a transcription bubble and you're going to form negative super coils um um behind the transcription bubble and again this is um remedy by the too isomer races gases for example are used in um relieving the positive super corals here and the Topo suas is used in relieving the negative super cles okay so there airas is an example of a topas so top suas removing negative super coil and a toomas here that is removing positive super coil now what do you think is going to happen if if and these are the questions that I want you to familiar with because these are the questions that I may ask you in the exam like um what is going to happen say for example a gas or Topo isomerase is dysfunctional such that it's not able to um relieve via the super coils that case what happens is that this will definitely what this will definitely um start I mean stop from happening the transcription because you cannot unwind a segment of the DNA indefinitely without um without relieving the super coils right the tension on the duplex will be too high after some time it won't unwind anymore all right the RNA polymerase catalyzes the formation of a fals diaster bonds I've already we've already mentioned that the tropis rases relax the super coils in front of and behind the transcription bubble okay now how does a process aimed how does a process aimed that is a very good question so there are two types of mechanisms for um the the termination of transcription one is called intrinsic termination it's also called row independent row independent um termination and the other one is row dependent termination it is um R is um A protein that um signals uh the end of transcription we're going to discuss that later for now look at that let's first look at the intrinsic termination so what happens is that in this case you form palen drones now you you you you I mean you encounter palindromes or palindromic sequences okay um a termination sites are characterized by two inverted repeats okay now what am I talking about so remember we have two DNA strands so this is your right this is your coding five Prime 3 Prime three prime five Prime is your um template strand okay all right so notice that there is an inverted repeat here and then there is another inverted repeat now what does that mean what are inverted repeats okay inverted repeats or inverted repeats are sequences of DNA that are um the same they're actually not the same how do I'm going to so um okay all right these are sequences of DNA that are the same when read in the same direction okay I think it is best uh demonstrated B it's example so H look at this a look at the uh laser pointer a a a g g c t c c okay and what do you notice here this is a a a g g c t c c oh and remember this is our coding strand it's all right it's from five Prime to three Prime and this is our template Str it's from five Prime to three prime and look at that it's actually what it's actually the same right when you read them in the same direction like from five Prime to three prime and here from five Prime to three prime a a g g c t c c that is and here you have a a a g g c t c c what do you mean they are the same right but here I have to be more technical because you're actually reading this let me use a pen um you're actually reading this um here in that direction going to an arrow there and here you're reading this in this direction okay and effectively when you read them in opposite direction they read the same right because this is read in that direction and that one is read in that direction but in Ence in principle it's just it's the same five Prime three prime five Prime three prime but for the purpose of of that definition um you have when you read this sequence from left to right and that sequence below here from right to left you read the same thing a a a c g c t c c a a a g g ctcc we call that um in inverted repeat all right or also a palindrome all right a palindrome now here is another inverted repeat like let's see what we have here this is g g a g c c TTT and look at that here it's also read the same g g a g c c t t t so that's another inverted repeat right okay another term for that is a palindrome it's um it's um sequence that when read in opposite direction goes right gives rise to the same sequence and U I'm not sure have you heard of a term palindrome before that is um because I want to uh emphasize the point let us um let me see I'm not sure if you have heard the term palindrome uh and you give me a palindrome in in there are also palindromic words right and I'm not sure if you have uh um encountered a pend Drome before because when you read race car what happens it's uh when you read it um from left to right it's race car when you read it from right to left it's also race car right yes here is another palindrome was it a cat I saw right when you read it from right to left it's also was it a cat I saw very interesting right I love T drums let me um um um here let's see is this a paland drone um no lemon no melon so when you read that from right to left it's also no lemon no melon remarkable right just one more because I love paland drums and I I want to emphasize the point but you have palindromic sequences there um um or maybe some palindromic words um let's see and they are simple palindromic words right um race car and this is one of the simplest one okay I'm going to this is one of the simplest B Dr here wow right that is the simplest one of the simplest palr okay now enough of palr let's go back to the palindromic sequences okay as I mentioned a while ago here we have inverted repeats or palindromes and this is very interesting because what happen is that okay this is your coding strand right this is your coding Strand and this is your template strand what happen is that when your RNA is synthesized you end up with this all right and what do you notice this inverted repeat is actually compx mentary to that inverted repeat and what do you notice what kind of structure is this this is a hair pin Loop right a hair pin Loop and this hair pin Loop um is important for the process of germination because as this is formed the um uh the transcription um stalls and ultimately the transcription terminates also notice that you have here a a a a a and so that means you have after that second inverted repeat you have u u u u u u remember Au just has two um hydrogen bonds between them and therefore the interaction is weaker that explains why when you have this palindrome encounter this palindromic sequences and you have TT T TT in the coding strand you are expecting that the process is going to be terminated because first you form a hair pin Loop that um ultimately that has the effect of slowing down and ultimately terminating the process of transcription and you have uuu which um um is which forms um weak hydrogen bonding with the template sand ultimately allowing the MRNA to um what do you call that to dissociate okay let's go back to that slide um as I've mentioned a while go the intrinsic termination does not require the activity of the row factor or row protein now in the in the intrinsic termination in the intrinsic termination the um the um the formation of a heroine Loop resulting from inverted repeats and the um polyu the polyu factor causing effectively cause the process of transcription to stall and ultimately terminate right the MRNA dissociating from the DNA the roow dependent termination requires or involves the activity or the participation of a row Factor it is quite small but I hope that you can still see that the row Factor against see here the r factor it's a protein right bad protein all right now notice that that you have here an RNA polymerase right and the RNA polymerase um and here is your transcription bubble your transcription bubble okay so the RNA polymerase um extends the RNA um and as it extends the RNA you see the row Factor actually trying to cut to catch up with it right it's actually trying to catch up with it so the row Factor mechanism of transcription termination the ru Factor attaches to a recognition site on mRNA it recognizes mRNA okay not the DNA the MRNA and moves it along behind the RNA polymerase in other words it's trying to catch up with the RNA polymerase when RNA polymerase pauses at the termination St remember why does it pause at the termination St um uh the U um it it PES or it slows down at the termination site because of the formation of that hair pin Loop okay so when RNA pimas poses of the termination site roow Factor unwinds the DNA RNA hybrid in the transcription bubble have here remember that's the DNA RNA transcript so the row factor is able to catch up with the RNA polymerase because the RNA poly stalls or slows down or paes at the uh at the uh termination side and as it unwinds the DNA RNA hybrid the um mRNA is effectively released or um liberated out of the DNA RNA hybrid and the process is effectively terminated or the process of transcription is effectively terminated okay now this um um again involves row dependent protein or a row Factor okay and of course you still have here the formation of Hain Loops okay now in procot there are different ways by which we regulate transcription which I've already mentioned we transcribe or proteins are transcribed I mean DNA are transcribed at differ at different um rates right some some genes are transcribed more often than others and some are not transcribe at all the junk DNA so in procaryotes how do trans or how is transcription regulated there are a number of different ways with which we do that the first is through alternative Sigma factors second is with the use of enhancers and then the oper Rons and then the transcription attenuation first discuss the alternative Sigma factors but is again Sigma if you remember Sigma um um Sigma if you remember um the sigma Sigma subunit is important it's an important subunit of the RNA polymer without which transcription cannot be initiated so let's go back to uh let's go back to U that um um um let's go back to that so one of the ways by which one of the ways by which um the um the uh procaryotes regulate transcription is through alternative Sigma factors the sigma factors of already mentioning over and over again they are important because they bind to a promoter region and the sigma factors have different binding affinities to different promoter sequences in the same way that different promoter sequences have different different binding affinities toward Sigma Sigma factors so viruses and bacteria exert control over which genes are expressed by producing different Sigma sub units that direct the RNA polymerases to different genes um I hope that this is clear so what happens here is that early transcription so for example if you have a virus which is infected you initially so what happens virus is actually dead in the when it's not um associated with the living host it's dead but once it becomes part of the living host it's it assumes properties are characteristics of living things so it's now alive in other words so the the early transcription what happens first is that the virus hijacks the uh transcriptional machinery of the cell or of its host so it uses the um um Sigma factor of the host the host Sigma subunit so and then it uses that so this is the gene right this is the gene of the virus so the early quote unquote genes of the virus are transcribed using the sigma subunit of the of the of the host Sigma subunit of the host and what is and you're going to Marvel at that V um virus the virus early genes that are actually expressed these are many but but one of them are the gp28 and what is gp28 that gp28 is a specificity factor in other words a sigma subunit all right a sigma subunit of the virus rather a sigma subunit of the RNA polymerase is that actually the RNA polymer of the of the virus but mostly the RNA polymerase of the host all right but this Sigma subunit is now the sigma sub unit that is specific for a particular region in the virus DNA or in the viral DNA okay gp28 and so it look at that first we're expressing this early genes now we're expressing this middle genes of the virus and this middle Gene of the virus you form the middle transcripts which include the um uh middle proteins gp33 and gp34 and gp33 and gp34 are other other Sigma subunits Sigma subunits that are important in recognizing the promoter region of the late genes of the virus so what do you notice as the process of transcription in this in the vital transcription occurs in this case the the um Sigma subunit of the virus become the predominant Sigma subunits right and it's no longer the uh Sigma subunit of the host that um determines or that that is um that determines um much of a transcription process but the sigma subunit of the virus that's how the virus um takes over subvert and um or subverts and um hijacks the transcript ctional Machinery of the host right viruses are well they're opportunistic okay all right now as I've mentioned in in instead of alternative Sigma Factor you can also have enhancers now what are enhancers enhancers are certain genes which include sequences Upstream of the extended promoter region and this enhancers as the name suggest enhanced transcription this genes for ribosomal production have three op stream sites fist sites all right the class of DNA sequence that do this are called enhancers they are they are bound to proteins orever they they are bound by proteins called transcription Factor so here is a bacterial promoter remember you've already encountered this before right so you have this core promoter region and you have the transcription start side here you have this what is this the prol box and you have this negative 35 another consensus sequence and you have here this is the region all right the up element and up element region and you have here the fist sites this fist sites one two three these are the enhancers all right certain proteins bind them to this F sites which um the the net effect excuse me is the enhancement of the process of transcription the process of transcription of of uh of the gene here right the gene here okay um by the way this up element and the core promoter consist what we call the extended promoter right the extended promoter as you can see here okay again this corresponds to the coding strand to five Prime to three prime strength okay again as I mentioned this fist sides of enhancer this become bound to proteins called transcriptional factors that um encourage The Binding of the RNA polymerase at the promoter region and therefore ultimately leads to that particular Gene product being transcribed more often and at a greater um speed okay G frequency now here is a very important topic called the operon now the operon is um again another way by which um the um procreates control the process of transcription the operon um corresponds to a group of of of Gene sequences all right you have here an operator right a group of operator promoter and structural genes with codes for protein right okay um the control sites as as uh specify here as specified here um um include the promoter and the operator genes all right these are the control sites of the operon don't worry we're going to discuss more of these in detail what I'm trying to say is that an operon is a group of of of of of DNA sequences which consists of an operator all right the promoter and the structural genes that codes or proteins this the structural genes primarily um are families of proteins that are involved in a particular metabolic pathway okay so don't worry we're going to deal with that touch on that in detail later the control sites here uh the control sites promoter and operator genes they are adjacent physically adjacent to each other um to the structural gene in the DNA right the control SES from alter and operator genes are physically adjacent to the structural gene the DNA the regulatory Gene can be quite far from the operon and the regulatory Gene can be quite far from the operon operons are usually not transcribed all the time they're typically not transcribed an example is the um Lac operon the lactose operon so the beta gcto is it's an inducible enzyme in other words it is typic Ally not expressed but you can induce it its expression in other words under certain conditions this substance can become expressed all right it is coded for by a Str a structural gene all right lack Z lack Z codes for the beta galactosides enzyme you know what this beta galacto you've encountered this before already in one of the lab activities remember the lactose lactose is hydrolized by Beta galactosidase into galactose and glucose okay so it is coded for by a structural gene called laxi there's also structural gene Lai coding for lactose permas it the lacto perous protein of course is important for the um transport of um transport of uh um um La transport of the um uh lactose into the cell all right and then the structural gene Lac a calls for Trans ayat all right the activity of trans acetat is not that clear but it is believed to be important in uh um neutralizing certain um not neutralizing at least interacting with certain antibiotics um the expression of this fre structural genes is controlled by the regulatory Gene called la eye codes for a repress okay now here is the um Lac operon okay hope that this is clear so again you have here the La Z right the Lai what is the LA Z code for the LA Z course codes for a repressor I'm sorry it codes for the beta galactoses la for the um lactose fmas and Lac a if you remember codes for Trans acetylates all right this um Gene products or this proteins Gene products are just proteins right so this Gene products or this proteins are important for the metabolism of lactose now a repressor protein is coded for by Lac I lack ey all right lack eye okay now I want you to notice that lack ey is actually physically close to the lack Z lack Y and lack a right so the lack eye codes for a depressor protein all right so that lack eye codes for leads to an mRNA transcript this one but ultimately codes for a repressor protein and this repressor protein forms a tetramer forms a tetramer all right and this tetramer all right binds to look at that but this Pac is actually the promoter region the promoter region for this lack Z lack Y and Lac a um so it binds to a region um called o it's the operator and notice that the operator actually overlaps I believe it overlaps with the promoter region okay so the operator and the promoter sites together are the control sites of this operon so what happens is that this repressor protein binds to that operator region and what does that mean this means that when this repressor protein is bound to the oper operator sequence or the operator region lack Z lack Y and lack a cannot be expressed why because there is a protein that prevents the RNA polymerase from binding to the promoter region right remarkable so that means that no beta galactosidase no lactose permas and no trans atilas are going to be formed or synthesized all right okay because of a repressor protein now we said that um in the presence of an inducer in the presence of an inducer lack Z lack Y and lack a are expressed in other words you form beta galactoses you form um um lactose spermes and you form trans atilas and what is this inducer this inducer is actually lactose right all right okay this inducer is actually called lactose when you say for example groad aser Shaka or groad bacteria in in a in a medium that contains lactose that that lactose all right binds to the repressor remember the repressor tetr of the repressor protein binds to the operator in the process preventing The Binding of the RNA polymerase and preventing Lac C Lacy and Lac a from being transcribed but in the presence of an inducer in this case lactose that um repressor protein binds to the lactose molecules effectively the activating the repressor the repressor can no longer bind to the operator sequence and therefore the RNA polymerase can bind to the promoter and in the process lead to transcription ultimately leading to the formation of beta galactosidase lactose permas and trans atilas and that is remarkable why because in the presence of lactose lactose needs to be metabolized and this proteins beta galacto um permas as well as transas are important for its metabolism okay this is the lack operon and notice that this is an inducible an inducible um Gene you induced its um expression by the presence of an inducer the lack toast the lactose all right okay now apparently and this is very remarkable apparently if you have lactose but you and you do not have glucose beta galactosidase permas and trans atilas are going to be formed but if you have lactose and you have glucose beta galactosidase permas and transacetylase will not be formed again again if you have lactose but you do not have gluc beta galactosidase spermes and transas will be for because the the bacteria does not have any choice but to metabolize lactose hydrolize it into glucose and galactose and use it as a source of nutrient but if you have lactose and glucose the bacteria will have no need right will have no need of expressing beta galacto sperm and trans at least because it has glucose to metabolize so in the presence of lactose and glucose lack Z lack Y and lack a are not expressed why is that the case okay apparently um Lac operon as I've mentioned all right is induced when eoli has lactose as the carbon Source Lac uh protein synthesis is repressed by glucose as I've mentioned if there is glucose the lack uh beta galactosidase the lactose permas and the and the um trans atilist won't be expressed or won't be formed because there is glucose and how does glucose do that process is called catabolite repression eoli recognizes the presence of glucose by promoter as it has two regions the RNA polymerase binding site and the catabolite activator protein binding site now remember this is the um what do you call this is the promoter right the uh promoter region of the um okay you remember that this is the promoter region all right the promoter region of the Lac operon apparently the promoter region of the Lac operon has an RNA polymerase binding site and you have your a capap what is Cap catabolite activator protein it has a catabolite activator protein binding s okay now um catabolite repression is another way by which procaryotes control transcription so the capap forms a complex with cyclic adenosine monophosphate now when you have a lot of cyclic adenosine monophosphate that means that um the all right let's do this step by step the control sites of a Lac operon the cyclic adenos uh I mean catabolite activator protein cyclic complex right not capap alone binds to a capap site on the Lac promoter right it's this C plus capap forming this complex that binds to the CM complex when the capap site in the promoter is not occupied RNA polymerase does not bind all right in other words when this all right when this um when when the C site on the promoter is not occupied RNA polymerase does not bind so this this has to be present in the promoter in order for the RNA polymerase to bind okay in the absence of glucose cyclic forms a complex with C the complex binds to the C site allowing RNA polymerase to bind to the entry side on the promoter and to transcribe the structural genes okay let's do this step by step we already know that in the presence of lactose lactose is an inducer remember it's going to lead to the formation of of the expression of lack Z lack um lack Y and lack a in other words the formation of the um beta galactoses the lactose permas as well as via um trans aetas but we know that in the presence of glucose this Gene products are not expressed so there's something happening here other than other than induc other than the induction by lactose and this is what we're talking about glucose all right in the in the absence of glucose when there is no glucose but there is lactose cyclic amp forms a complex with cap right because in the absence of of glucose the absence of glucose the asakai is starving it is starving all right means it does not have any choice but to um Express the galactosidase lactose permat and transacetylase in order to hydroly in order to take in lactose and hydroly it into galactose and glucose as a source of energy as a source of carbon so when when there's no glucose cyclic amp is high all right because that corresponds to a starved State cyclicamp then binds to the catabolite activator all right remember that catabolite activator protein capap and that binds I that forms a cap Camp CM complex and that complex binds to the cap side allowing RNA polymerase to bind apparently this is needed for the RNA polymerase the PIP okay that is why in the absence of glucose there's a lot of CM the cell is star and so the CM forms a complex with capap which CA capap C complex and binds to the to the um to The capap Binding site in the promoter region which allows her RNA polymerase to bind to it and therefore beta galactosidase um lactose spmat and transas are expressed because the cell needs those at least to lactose permas as well as the beta galactosidase in order to have glucose from the lactose as its energy source okay now why is it that when you have glucose lactose permas transacetylase and um beta galactosidase are not expressed it is because when you have a lot of glucose the cell is not starved and therefore you have you have have very few cyclic very few cyclic so that means the capap CM complex is very very low in concentration remember it is this complex look at my look at my uh pointer it is this complex that binds to The capap Binding site in the promoter region not the cap so that means that here what happens is that you do not have capc M complex nothing binds to the cap binding site in the promoter region and remember this cap CMP complex is important for The Binding of the RNA polymerase without it the RNA polymerase cannot bind and therefore the beta galactosides the lactose spermes this Gene products won't be expressed and it makes sense because you already have glucose you don't need to express beta caloes all right this is an example of catabolite repression okay now as we mentioned the Lac Opera the lactose operon is an example of of um of um a negative control where you have a repressor that binds here and preventing RNA polymerase from binding but in the presence of of a a co-inducer like lactose that inactivates the repressor and so the MRNA then um is is allowed to be synthesized in the case of catabolite repression you have an IND inactive inducer that inactive inducer all right um becomes activated in the presence of cyclic amp all right and you it binds here in the in the uh promoter region so whereas the lactose operon gives you negative control the catabolite repression is a positive control all right okay in the case of lactus operon the repressor deletions are constitutive in other words when there's no repressor the um Gene product is being expressed constitutively in other words it's just being expressed like form and form and form of that you form and form and form more and more of a gene products in the case of inducer deletion when you do not have the inducer right the active inducer you do not express the genes Downstream in other words it is uninducible all right now let us consider V tryptophan operon there's also a tryptophan operon all right here tryptophan operon um okay now the trypan operon codes for a leader sequence trip L and five polypeptides so lber is trip L here is a trip L you have trip e trip D trip C trip B and trip a now this um enzymes are actually important in the conversion of chorismate to tryptophan right cism to tryptophan in other words the metabolism of tryptophan so cism made to tryptophan now the trip L is um the leader sequence the five proteins trip e trip D trip C trip B and trip a make up the four different enzymes catalyze the multi-step process with converts coris Mage to tryp toine okay now um what happens here is that all right let us go back here so you have a trip to find operon all right in this case there is a an inactive repressor so your repressor is inactive in the presence of a COR repressor it becomes activated and you have an active repressor so here it's it's different in the case of the Lac operon because in the case of a lactose operon your repressor is already active right it's already active and you need um lactose in order to deactivate it in order to for it to be inactive in this case your um repressor in the case of Tran opon is inactive you need something to activate it and that something is actually what that something is actually trypto all right okay that something is actually tryptophan let me um go back to this this is the trypt toine Opera right and you have here the trip toan operon so what happened is that this Gene trip e trip D trip C trip e and trip a are needed for the synesis of tryptophan if you already have a lot of tryptophan do you think you still need to ciz tryptophan no right because you already have a lot of tryptophan and so what happens is that excuse me what happens is that if you have a lot of tryptophan the tryptophan binds to the repressor which is expressed in the tryptophan operon all right and where is that here is a tryptophan operon all right so this tryptophan operon cises um a repressor that repressor is inactive but when it binds to tryptophan it becomes activated so when it when it binds a tryptophan it binds to the operator and therefore prevents as you can see here prevents the transcription and it makes sense because if you have a lot of tryptophan and tryptophan is your core repressor that tryptophan will buy into the inactive repressor causing it to become active if you have a lot of tryptophan you will have a lot of active repressor and therefore will prevent the expression of the genes necessary for the CIS of tryptophan and it makes sense right you already have a lot of tryptophan you don't need the the genes or the enzymes necessary for the synthesis of trypt ofine okay all right okay I hope that that is clear now um um you will remember that we have here um a leader sequence trip L all right what what does that excuse me what does that trip L do that trip L leads to the formation I mean that um leads to attenuation all right in the trip Define operon okay let us consider all right now I'm trying to uh see how to best explain this to you okay now so let's look here these are the alternative secondary structures that can form in tripto find operon so this structures can form in the leader sequence remember you have here a leader sequence right that is a leader sequence that preds trp toine e trip toine d trp toine c trp toine b and trp toine a um Gene Gene products okay this structures can form in the leader sequence all right all right look at that what do you notice these are hair these are all these are hair pin Loops all right so you have here one then you have here another and then you have here another now these are po structure blinding between po structure these are the binding between regions one and two there's a Terminator Loop binding between regions three and four all right now we haven't yet discussed the process of translation but I want you to um bear in mind that the process of translation and transcription in procaryotes are not separated but is as the MRNA transcript is being synthesized as mRNA transcript is being synthesized you're forming or the Mr is being translated into protein at the same time that is different from ukots like in humans for example because in in the case of of humans the the MRNA transcript is being Sy is first synthesized and then it is transported into the site to Sol for the process of translation here it's not the same in procaryotes because in procaryotes transcription and translation happen at the same time so these are the alternative second D structure that con form in the trip toine offer as I mentioned this structure is can form in the leader sequence and what do you notice here are tryptophan codons all right here are the tryptophan codons tropon trp ofun okay and these are very important because um these are the uh this will be very important later when in in the next slide when we discuss the other mechanism of of procaryotic transcriptional recogn uh regulation okay as I've been mentioning Paul structure so this is Paul structure one two and this is a Terminator um region 34 and notice that you have here a PA structure and you have a Terminator region okay and what do you remember of a Terminator region once again it is rich in U all right as before and you have this um hair pin Loop okay now another another U mechanism for um transcriptional regulation in procaryotes is the attenuation so it happens when the um process of transcription is slowed down after it's started so the pole structure forms when ribosome passes over tryptophan codons when tryptophan levels are high so this is the ribosome all right this is the ribosome and remember if a ribosome translates Thea um protein all right the trans the ribosome translates the protein so the ribosome here is transcribing the leader peptide mRNA strand so when you have high tryptophan the um RNA polymerase is um terminated why because okay remember you have here tryptophan codons right this tripop codons will be um recognized by the trnas the trnas that are charg with tryptophan right that are charged with tryptophan such will cause the ribosome to trans late the uh the uh that region with high trypt ofine and such will cause Thea um the RNA polymerase to pause and ultimately terminate the pause structure forms when ribosome passes over tryptophan codons when the tryptophan levels are high as we've already mentioned the ribosome stalls the tryptophan codon all right when tryptophan levels are low and anti-terminator loop form so here is when you have high tryptophan okay and here is when you have low tryptophan you form here an anti-terminator Loop and what happens is that the ribosome stalls right when when there's you know what what happens is when when you have low tryptophan there are of course less trnas that are charged with tryptophan so the process of translation will therefore be slow and so the ribosome stalls and you form here an anti-terminator Loop which allows Thea um Thea RNA po to continue transcribing the um the uh um tripto find genes trip e trip D trip C trip B and trip a again necessary for the cenes of tropine for from coris because you have low levels of of uh of uh of uh trip toine all right all right so so far we have studied the we have studied the transcription in procaryotes those are a transcription in procaryotes again how does the procaryotic transcription differ from eukariotic transcription the fact is that um procaryotic transcription occurs at the same time as the um procaryotic translation as you can see here the protein product is being formed at the same time that the MRNA is being synthesized the process of transcription transcription and translation occur at the same time the same is not true in the case of ukots all right case of ukots there are different RNA polymerases RNA polymerase one two and three RNA pimmer two is primarily found in the nucleoplasm and synthesizes the MRNA precursors all right and in the case of ukots the transcription occurs inside the nucleus once a transcription is that the MRNA transcript is transported into the cytool for the process of translation okay now here you have a number of different um transcription factors all right but the process is essentially the same as in the process of transcription in procaryotes that's why we first discuss the transcription in procaryotes so here instead of you have again um the promoter sequence the promoter sequence you have uh remember in the in the case of the procot you have a prno box here you have a Tata box or you have a Tata box I think unless I'm mistaken this is also called the hgess or hgess um box in any case that's a Tata box and that's the promoter region and you have here the initiation start side plus one again this is the coding region all right the coding region okay now and then of course of course The Binding of the transcription factors are very important as okay here is um the order of events in the transcription in transcription in ukar first and foremost less is known about the transcription in ukots and procot that's why we first the cost transcription in procaryotes but a similar thing is believed to be happening but the only difference is the details it's more involved in the case of uh transcription in ukra so first is the recognition of this data box the promoter sequence afterwards you have this um um the you have this um tfid um binding this is a transcription factor and then afterwards what it recruits other transcription factors like protein B um here are they all right this is a tfid because you're wondering sir what is tfid TFI D is a tatab box recognition um positioning datab box recognition protein it positions that it positions via um um it's necessary for positioning of a tatab box DNA around tf2b and polymerase 2 these are simply the general transcription initiation factors these are needed for the initiation of the transcription process and as I've mentioned there are so much more proteins involved in eukariotic transcription compared to procaryotic transcription the principle is just the same so um what you have here is tfid D2 binding to the tab box and then other proteins are recruited until ultimately you recruit polymerase 2 RNA polymerase 2 what happens is you recruit more of a transcription factors and you form this pre-initiation complex formation I mean you form this preinitiation comp complex all right all right now I want you to notice that in this case what happens the DNA is still closed right it is still closed but here the DNA stands become separated so you have an open complex form then afterwards the polymerase 2 is phosphorated by a kinase this is polymerase right look at this that polymerase does not or is not phosphor yet after it is phosphorated all right you form this phosphorated um um polymerase 2 and after the phosphorilation you notice with the duplex DNA forms all right okay when the polymerase is um phosphorated when the process of transcription occurs so you form that mRNA transcript this is the green one the green one here is the MRNA transcript and so on so forth until the process is terminated and when it is terminated the polymerase 2 is simply Def phosphorated and a gain recycle gain recycle all right um as in the same way that the initiation of transcription is controlled by um transcription factors the process of elongation is also controlled by a number of different transcription factors all right like positive transcription elongation factor and negative transcription elongation Factor the truth is the process of UK carotic transcription requires a lot of of transcription factors or transcription proteins um termination Begins by stopping RNA poies the eukariotic consensus sequence for termination is a uaaa again this is a rich and you rich because Thea Au forms weaker hydrogen bonds just two hydrogen bonds in comparison to GC okay all right again the consensus sequence is a uaaa when you have that region then that signals the RNA polymer to stop stop transcribing as in the case of uh procaryotic um procaryotic systems or procaryotes there are also enhancers like enhancers um but increase the um frequency of transcription there are also silencers these are proteins that actually decrease the frequency of of uh of the transcription so the DNA looping because the enhancer can actually very very far from the from the promoter as well as from from the gene itself so the DNA looping brings enhancers into contact with transcription factors and polymer so see here the enhancer is quite far but the DNA can be looped upon itself and it brings tfid D2 RNA polymer is too close to the gene product to the promoter the gene that you want to express right this is Gene regulation in ukar okay um the response elements are these are just enhancers that responds to certain metabolic factors like heat shock elements glucocorticoid response element the metal response element the cyclic response elements this the the principle behind this is relative simple they just um these elements become bound to particular proteins which then um enhance the expression of say for example heat shock proteins and those that are um associated with the glucocorticoid response for example all right and there are of course this response elements in their characteristics you have a consensus sequence for example for CR for the hsse the heat shock all right um You have this consensus sequence for that they have their specific consensus sequence for this kind of enhancers all right now this is what I want to emphasize as much of as much as 98% of transrational output from un genos may be comprised of non-coding rnas okay in the case of procot the RNA is RNA produces translated only almost at the same time we're almost in Synergy as the process of translation it is being RNA is being um translated at the same time that the DNA is being transcribed right and so we say that perhaps most if not the entire stretch of the messenger RNA is actually translated in procot but in the case of UK carot as much as 98% as much as 90% of the MRNA transcript is actually not translated and that is that is interesting right that is very interesting you know what because um um let us see I hope it's here um because the transcript the the messenger RNA transcript is actually modified it is post transcriptionally modified all right um let me see here the modification of the M name I'm looking for introns and exons all right I all right this is what I'm looking for the the exons and the intron the exons are the part of the MRNA that are actually translated okay so this is the DNA coding strand all right and it codes for what we call exons and introns the introns are the DNA that are not I mean the DNA region that are transcribed but are not translated so look at that you have this Gene product and you end up with this mRNA transcript consisting of Exon Exon as well as intron the green is the intron the green is the intron the green is the intron so you have exons that are in purple no but this intron are not translated because they the MRNA transcript is processed it is process post transcriptional processing causes the intron to be removed and you end up with a mature mRNA okay and it is this mature mRNA but is translated and notice that it is not only that it is post instructionally processed such that you end up with methyl guanosine or mg cap and you have po a tail all right includes capping of the five Prime n with an N methylated guanine all right so here you have you cap it with an N methylated this is methyl group can see the methyl group here and methylated guanine all right includes um the capping of the five Prime n with an N methylated bonding that is bonded to the next residue by five Prime to five Prime triphosphate this is remember F Prime and this is another FIB Prime so it is f Prime to F Prime triphosphate and you have here um methyl guani all right the mature mRNA that is ultimately um ultimately um translated is or has a f Prime methyl guanine Cap all right and also it is poly aenul a tail that is usually 100 to 200 nucleotides long is added to the free Prime end before the MRNA leaves the nucleus as you can see here this is the poly aenul tail and you have here a seven methyl guanine cap and it is this mRNA that is transported into the cytool and which is ultimately translated as I mentioned the the Inon this introns are removed and that entrons are removed by the process called splicing all right what happens the exons are separated by intervening regions called intron so Exon Exon intron so what happens is that the system or the MRNA removes the Inon through this process okay uh Exon so you have here Exon so you have this two prime hydroxy group of an intron attacks this region okay in the process forming this sequence okay and what happens next is that this Exon becomes connected to the other Exon I'm not sure if you can see that so you have this is Exon one right and you have five Prime n so what happens is that it just bends upon itself follow my my my my pointer so it just bends upon itself that way I hope you can see that so it bends upon itself that way so now that it is bent that way see you have here the branch side and the splice side so you you splice it out all right this um this Exon is spliced out and this this Exon is ultimately um bound to another Exon in the process you end up with Exon being bonded to another Exon okay and you remove the the intron not when the exons are spliced together aarat forms in the Inon this is a laat structure okay so the point that I'm trying to um get across is that the process of transcription in OTS there similarity to the process of transcription in procaryotes but it is much much much much more complicated in the sense that it involves a number of different transcription factors transcription Factor protein that are involved in in initiation in elongation as well as in um well not much in termination but uh they're primarily in in in initiation as well as um elongation of the MRNA transcrip and not only that once the MRNA transcript is formed certain regions are spliced out the intron are removed and you just combine or you just end up with exons the express regions that are are that are spliced together and also the the um the five Prime end is capped with a methyl guani and Thea three prime end is labeled with a poly adenol tail right and then ultimately transported into the cytool for the process of translation all right now um that is a that's uh all that I want you to uh bear in mind and to um uh um uh to bear in mind and and study for the process of transcription