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Lecture 9

BIO240H Lecture 9.doc

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University of Toronto St. George
Jennifer Harris

Lecture 9: Transcription in eukaryotes Lecture Outline: 1) Structure of a gene 2) RNA polymerases 3) Transcription of protein-coding genes 4) RNA processing Readings: Alberts, Ch 6, pp.339-353 Slide 1 DNA Primary RNA transcript RNA cap - Gene structure is more complex in eucaryotes. - In eucaryotes: genomic sequences (coding sequences in genomic DNA) are interrupted by introns – intron sequences are transcribed to form mRNA – further processing has to happen to primary mRNA transcript in order to delete these intron sequences in order to make mature mRNA transcript. - 1 of those things are removal of intron sequences to put together entire coding sequence of protein. Other things that need to happen is putting on of RNA cap at 5’ end (5’ capping) as well as poly-Adenylation at 3’ end. - Then you can have exporting of mature mRNA transcript out of nucleus & into cytoplasm where it can then be translated – something that doesn’t happen in prokaryotes. This is much different than in prokaryotes which only have direct transcription and translation. Slide 2 - Eucaryotes have 3 different kinds of RNA polymerases not just 1 as the 1 found in procaryotic – tend to transcribe different sort of genes. - RNA polymerase II – transcribes protein coding genes – this is the most similar to the polymerase in procaryotes. 5.8S, 18S are different sizes of the mRNA Svedberg units are the measurement of RNA sizes There are a distribution of jobs as in three different RNA polymerases which specialize in their own synthesis. Slide 3  Bacterial RNAP’s - Eucaryotic RNA polymerases – lots of these subunits are actually homologous to each other – thought to be derived from similar ancestral sequences. Across 3 different polymerases, they share some subunits in common, but then have some subunits that are homologous to each other, but not exactly same, not encoded for by exactly same gene. - RNA polymerase II resembles most closely to E. coli RNA polymerase – most similar, thought to be homologous. There are more subunits found in the Eucaryotic polymerases There are beta-like subunits, alpha-like subunits, omega-like subunits as well for all three Common subunits are all found between the three There are also additional enzyme specific subunits Since they’re similar, this indicates that they most likely divulged from the same ancestor at some time during evolution Slide 4 - Eucaryotic has additional domains (subunits that are part of hollow enzyme that perform supporting roles). - 3D structure is exactly the same. 5 different subunits even in very simple polymerases in prokaryotes (Ex. E coli) Substructures that are the same are colour coded (similar at least) The colored regions are fairly homologous All eucaryotic polymerases look similar to the prokaryotic polymerase but polymerase II is most similar, eucaryotic one has extra domains Polymerases between species is highly conserved Slide 5 1. Transcription factors - Promoter is sequence in genomic DNA that actually helps position & initiate the transcription. There can be many. 2. Sigma subunit - Instead of being a subunit of the polymerase, these are actually completely different proteins and often, many more than one transcription factor is required in eucaryotes in order to initiate transcription. Reasons for whole host of factors needed for initiation of transcription – more complexities & gene regulation is needed in eucaryotes than procaryotes. 3. Chromosomal structure Refers to the idea that DNA in eucaryotes are wrapped around histones which forms the chromosomes. The histone proteins makes it a bit trickier to get at (DNA that needs transcribing) Harder to access DNA relative to bacterial DNA. Slide 6 Multiple Proteins 5’ Cap Poly A Tail - In procaryotes, level of primary transcript (mRNA transcript) can actually have different genes encoded in single mRNA transcript – those genes can code for multiple proteins – those proteins are separate proteins that can be involved in completely separate things – this is not usually found (very rarely) in eucaryotes – usually have single coding sequence that codes for single gene. - Exons & introns are not shown on this diagram – already has intron sequences spliced out, 5’ cap & 3’ poly-A tail put in – mature mRNA transcript. - These modifications of 5’ & 3’ end are not in procaryotic transcripts. - This idea that you need many different factors involved in splicing, modifying 2 ends – these are functions that actually happen during transcription. mRNA in prokaryotes are formed and can code for more than one protein We can see interesting features where there is the 5’ and 3’ end. The 5’ end has an untranslated region where start codon exists Start codons are found between the proteins and stop codon at the 3’ end The non coding sequences are only buffers between different genes in the mRNA transcript, it is no longer a primary transcript Slide 7  RNA processing proteins (capping factors, splicing factors, 3’ end processing factors) - The modifications are happening while the RNA polymerase is actually doing its job. As nascent RNA transcript is emerging from RNA polymerase, have factors that jump off & associate themselves with growing mRNA transcript & starting modifying already – very efficient. - Not all of these factors are associated at 1 time – some serve as nucleation sites to attract other factors that are not associated with RNA polymerase itself. - The factors associated with the RNA polymerase are associated with the C terminal tail/domain (CTD) of the polymerase. The phosphorylation of this C terminal domain of RNA polymerase II is what results in the binding of these RNA processing proteins (splicing factors, capping factors, polyadenalation factors). If it was unphosphorylated then the factors would not bind. RNA polymerase made up of many different subunits The carboxy means carboxyl end (carboxyl terminal domain) with many repetitive AA sequences Carboxy terminal domain is hanging off the carboxy terminal it is not a domain of its own This is like a RNA factory As it transcribes, it brings with it whatever it needs to do the RNA processing Slide 8 - We start with Promoters Slide 9 - Idea of promoter sequence in eucaryotes is similar to consensus Positions RNAP II binding sites for promoters in procaryotes for sigma factors.  Highly transcribed genes - There are sequences that tend to be preferentially recognized by certain transcription factors – there is some variation in those consensus sequences for a given transcription factor but even though there is variation, there are actually preferred nucleotides over others. - TATA box – 1 of most common & highly used promoters – generally found just upstream of the start site for transcription & is known to play a major role in helping to position the RNA polymerase II – tends to be found in the most highly transcribed genes – very strong promoter sequence. Highly transcribed genes don’t just have TATA boxes, they would tend to have several transcription factors associated with origin of transcription but TATA boxes are 1 of the most important ones. - Transcription factor associated with TATA box is TATA binding protein (TBP) – when it binds, it tends to introduce kink into the DNA which tends to loosen some of base pairs around this kink – induces conformational change in DNA which helps rest of what needs to happens for transcription. TBP essential for binding of other factors at the site of transcription initiation (including RNA polymerase II) as well as helping helicase for the separation of the DNA strands. You need to have a sequence on the 5’ or nearby end of the gene to signal start of transcription The general transcription factors will bind first and then RNA polymerase will bind In eucaryotic genes, there are the four that have been identified found in slide of chart Look at where transcription starts and those approximate regions of the promoter sequences INR starts over transcription start area and DPE is further down These promoter areas attract the factors which then attracts RNA polymerase TATA box is good attractor for general transcription factors Slide 10 Slide 11 - Each transcription factor has a different role. - There are differences in how these transcription factors assemble among different organisms. In most eucaryotic systems we do know about though, these transcription factors are the key players in the initiation of transcription. - TATA box is actually part of larger transcription factor TFIID transcription factor – it is binding of that one that enables the binding of other transcription factors & the loading of the RNA polymerase II onto the mature mRNA transcript. - It is the recognition of the specific DNA sequences (most important by the TATA box) that determines the specificity of the transcription initiation site. - There are many other components that are involved in different things. - TFIIB binds next, recognizes BRE element in promoters & it actually is 1 of major players in helping position RNA polymerase. - TFIIH – both helicase & kinase (2 separate domains). Helicase function helps separate the strands – uses ATP. Kinase portion phosphorylates the RNA polymerase in its C terminal domain and allows for entering of elongation phase & is essential step for all RNA binding process components. - TFIIE helps load the helicase, etc. TF stands for transcription factor and II refers to the fact that it is a general transcription factor for RNA polymerase II. BHC etc is for proteins with different functions For TATA box, TBP protein binds first which then attracts other factors Eventually you’ll get the start of the transcription factor RNA transcription finally starts Slide 12 1. TBP, TFIID - This TBP is recognizing that specific structure of the minor groove which exists even when the DNA is wound up around histones. TBP binds in the minor groove and bends the DNA allowing RNA polymerase to recognize the bend and initiates all of transcription. 2. RNA polymerase II (RNAP II) 3. TFIIH 4. Phosphorylates - RNA processing proteins can’t associate with C terminal tail if phosphorylation of the C terminal tail hasn’t happened. It is also necessary for the elongation conformation/phase of the RNA polymerase. - The key ones to know are TFIID & TFIIH Slide 13 2) 26 3) 52 - Mostly serines get phosphorylated: the phosphorylation of differen
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