Biochemistry 401 Lecture 37. Today we'regoing to talk about transcription, both in bacterial and eukaryotic systems.Hi, today we're going to talk about transcription. This is the synthesis of RNA, through the polymerization of ribonucleoside triphosphates by using DNA as a template. Before we get started, it's important to get some terminology straight, so that we all start on thesame page. RNA is transcribed from genes. When the genetic sequence is written with two strands, the top strand will be the coding strand and the bottom strand is the template strand. The top strand is called the coding strand,because it's the same sequence as the primary RNA transcript is going to be, except that the RNA is going to include U's instead of T's. The bottom strand is the strand that's going to be used for the template for the synthesis of RNA.We also have to have some terminology so that we can talk about directions in relation to a landmark. In transcription, there directions are upstream, which is in the five prime direction and downstream, which is in the three prime direction, and this is in relation to a landmark, andit's also in relation to the top strand. Why? Because in general, the top strand the coding strand is the more important sequence. For instance, when we write a genetic sequence and only use one strand, we write the coding strand, 5 prime to 3 prime. We don't write the template strand, generally speaking, and so when we're talking about terminology that includes upstream and downstream, we're talking about the five prime direction, and the three prime direction, respectively, in relation to the coding strand – the top strand. I hope that's clear.There are three main stages of RNA synthesis – initiation elongation and termination. Initiation is beginning, and in order to begin transcription, the polymerase, the enzyme that's going to synthesize this RNA, has to find out where to start. So it has to find a region in the DNA that's called the promoter. It's going to bind to the promoter in order to initiate transcription. So, promoter recognition, and then we have toform an open promoter that is melting the DNA strands so that initiation canproceed. in order to begin transcription, the polymerase actually has to leave thepromoter so that's called promoter escape. And then we have elongation, andfinally we have termination, when RNA synthesis stops. And so that's initiation,elongation, and termination. We're going to look at the synthesis of RNA in prokaryotes like the bacteria E .coli, and also in eukaryotes, and we're going to use humans as a model system for that.There are similarities and there are differences in these two types of transcription those in prokaryotes like bacteria and those in eukaryotes, like humans. The similarities for both systemsare shown here. Both required template DNA that have promoter and terminationsignals. they both require Ribonucleoside triphosphates, and these areAGC and U. We have U's instead of T's, remember. And polymerases in both systemsrequire magnesium as a cofactor. And finally, both systems require apolymerase but what isn't needed is a primer. Primers are not needed. Since we're using RNA for transcription anyway, we don't need an RNA primer. The actualprocess of polymerizing this RNA is also similar in prokaryotes and eukaryotes, inthis respect. The template strand is going to be read 3 prime to 5 prime andthe RNA itself is going to be synthesized 5 prime to 3 prime in a fashion that's complementary to the template. The synthesis of this RNA is going to be driven forward by loss of pyrophosphate and subsequent hydrolysis of the pyrophosphate to 2 molecules of inorganic phosphate is the same in prokaryotes and in eukaryotes. And another thing is, is that we needmagnesium in order to position these substrates at the right place inside thepolymerase and also to help catalyze the reaction itself. So magnesium is required as a cofactor.But there are many differences, too. The polymerases that are used in prokaryotes and in eukaryotes are different. For instance, there's one main polymerase for transcription in bacteria,but there are three main polymerases that are used in eukaryotes – Pol I, Pol II, and Pol III. We'll talk about those later. So the polymerases are different. Theway that RNA synthesis is initiated is also different. It's much more complex ineukaryotes than it is in bacteria, and termination is also different. Eukaryotesalso use a lot of post-transcriptional modifications in their RNA, especiallymessenger RNA. That's the RNA that we use to make proteins. That's substantiallymodified. We'll look at that in a minute, too. And another thing that's different, is thattranscription happens in the same place as translation in prokaryotes, likebacteria and archaea. That's because the genomic DNA is situated in the cytosol.There's no intervening nuclear envelope. And so, the transcription and translationcan happen at the same time on the same transcript. As a transcript's being made,it can also be translated. That's different in eukaryotes. In eukaryotes, our genomic DNA is in the nucleus, and so we can synthesize messenger RNA forinstance, but then that transcript has to be exported out of the nucleus, and intothe cytosol for translation. So let's talk about transcription in E. coli. This is a prokaryotic model system. And let's look at the polymerase first. This is the enzyme that's going to actually synthesize the RNA. This enzymecontains two major parts. It contains a targeting protein that is called the sigma factor.This is going to target the holoenzyme to the promoter. And it also contains thecore polymerase. This is the portion of the enzyme that's actually going tocatalyze the polymerization itself. Since the sigma factor is the "targeter", thisis going to provide the specificity of binding for the polymerase to actuallyfind the right promoter, to transcribe the right gene at the right time. And soE.coli has one main RNA polymerase, but it contains several sigma factors, andit's the sigma factor that provides the specificity for transcription forbacteria. The core polymerase is made up of five main regions – alpha 1, alpha 2,beta, beta prime, and omega. We're not quite sure what omega does, but alpha 1and alpha 2 are structural. Beta and beta prime are catalytic. Those subunits aregoing to be the ones that are actually going to catalyze the polymerizationitself. These two round objects that we see on the alpha
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