Biochemistry 401 Lecture 37 Today we re going 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 the same 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 and it 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 to form an open promoter that is melting the DNA strands so that initiation can proceed in order to begin transcription the polymerase actually has to leave the promoter so that s called promoter escape And then we have elongation and finally 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 systems are shown here Both required template DNA that have promoter and termination signals they both require Ribonucleoside triphosphates and these are AGC and U We have U s instead of T s remember And polymerases in both systems require magnesium as a cofactor And finally both systems require a polymerase 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 actual process of polymerizing this RNA is also similar in prokaryotes and eukaryotes in this respect The template strand is going to be read 3 prime to 5 prime and the 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 need magnesium in order to position these substrates at the right place inside the polymerase 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 The way that RNA synthesis is initiated is also different It s much more complex in eukaryotes than it is in bacteria and termination is also different Eukaryotes also use a lot of post transcriptional modifications in their RNA especially messenger RNA That s the RNA that we use to make proteins That s substantially modified We ll look at that in a minute too And another thing that s different is that transcription happens in the same place as translation in prokaryotes like bacteria and archaea That s because the genomic DNA is situated in the cytosol There s no intervening nuclear envelope And so the transcription and translation can 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 for instance but then that transcript has to be exported out of the nucleus and into the 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 enzyme contains 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 the core polymerase This is the portion of the enzyme that s actually going to catalyze the polymerization itself Since the sigma factor is the targeter this is going to provide the specificity of binding for the polymerase to actually find the right promoter to transcribe the right gene at the right time And so E coli has one main RNA polymerase but it contains several sigma factors and it s the sigma factor that provides the specificity for transcription for bacteria 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 1 and alpha 2 are structural Beta and beta prime are catalytic Those subunits are going to be the ones that are actually going to catalyze the polymerization itself These two round objects that we see on the alpha subunits are the CTDs the c terminal domains These are very important for stabilizing the polymerase on the
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