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Lecture 13

BIO1140 Lecture 13: Transcription (continued)

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Caroline Petit- Turcotte

Transcription (continuation) We are going to look at every step of this process (see video) -Transcription factors: proteins interacting with sequences on DNA, recruit polymerase to initiate transcription -polymerase joins in to the area of transcription factors, additional proteins that came and assembled and completed the complex of proteins and enzymes Start from DNA: to take sequences, the genes, and make a copy of the gene, but not an exact copy, we will be using RNA and prokaryotes, pre- -eukaryotes: mature RNA right away -which strand will be used? Which strand will RNA polymerase use to read nucleotides? 5’ to 3’. mRNA direction we will read amino acids to build 5’ to 3’, we need to read the 3’ to 5’ to make complimentary 5’ to 3’ RNA polymerase overlaps on both strands, complimentary from the 3’ to 5’ Prokaryotes: what’s the role of the sigma factor? The initiating protein prompter for prokaryotes. It also begins it in the right position (ideally); the first protein in the coding. -~25–40 before it’s released. Once RNA polymerase is stable, sigma factor will dissociate -helps stabilize and position RNA polymerase so it transcribes at the proper position -what happens if it’s too early of late? It would be the wrong DNA -important to position it at the +1, first nucleotide in the coding region In eukaryotes, it’s more complex; there are more proteins. The equivalent of the sigma factor TADA box binding protein. A series of Ts and As and it is recognised by this proteins. You can have extra binding factors that assemble along with binding protein, serve to recruit RNA polymerase II; place it correctly and initiate transcription -combination of proteins are different -TADA binding -factors -polymerase and can modulate -activators or repressors. On/off switch: you either start or interrupt transcription -dimmer effect—can have it occur: -slowly or faster; for a short period of time or even longer -all the proteins are the transcription imitation complex Transcription initiation complex -TADA interacts w/ consensus sequence; allows biding in other transcription factors -transcription factors: PKA, Krebs and cAMP, AP1 by kinase, Elongation -prokaryotes have one RNA polymerase and eukaryotes have more (specific, dependemt on RNA to produce) -the process itself of separating the DNA, reading and making complimentary RNA -the polymerase races down the DNA quick and adds 20 nucleotides/second -they make errors; but they are able to correct them in the DNA polymerase, however, not the RNA polymerase II Provide 1 key difference between Prokaryotes and Eukaryotes regarding termination of transcription The termination of transcription in eukaryotes can be terminated by a poly(A) signal along with a downstream terminator sequence, while the prokaryotic termination is completed by a rho-independent or rho-dependent mechanism. Prokaryote termination: 2 possible end-sequences Rho-independent mechanisms; forces them to detach from the RNA; it’s important that this is done at the end Rho-dependent (recognises sequences at the end_: interacts with DNA and prevents the polymerase from continuing Can have GC rich reasons near the end; one uses Rho and the other does not. mRNA is mature Eukaryotes: -a sequence at the end of the gene that serves as a signal; a poly A signal; tells the polymerase that it’s done transcribing—and recruits the Poly A polymerase Poly A polymerase comes in and adds an additional stretch to the RNA (pre-mature mRNA) -must be processed before it can leave the nucleus -once it’s finished being transcribed, the poly A tail is added at the end (50–200 adenines at the end) -mRNA is in the nucleus and it’s still not ready to end -once there are ~50 transcribed nucleotides, the 5’ cap is added, with 7 Gs (methalated, phosphorylated), and these are crucial for the mRNA to be functional (otherwise no translation) -happens after 50 nucleotides have been transcribed -the poly A tail added at the 3’ end is important and w/o it, the sequence cannot leave the nucleus -then the pre-messenger RNA is still in the nucleus with its introns and the objective is to get a continuous coding section—only exons -how must it make its way from the nucleus -diffuse through the nuclear pore, GTP dependent proteins that act as anchors, ensure that the Poly A tail is entact and shuttled across nuclear pores to the cytoplasm -Some will go to the ER and some will meet up with free-floating ribosomes in the cytosol Mechanism to splice mRNA -keep the 5’ cap, poly A tail, exons -rid the non-coding introns -will need some help in the nucleus -what recognisees introns vs. extrons -enzyme spliceosome contains enzymes, RNAs and SNERPS -SNERPS are proteins that are attached with SMALL NUCLEAR RNA ( -others are responsible for cleaving and ligasing the nucleotides together - to have these rnas that can recognise sequences that are at the begging/end of the introns -get rnas (they match) -short sections of double strand rna, helps recognise and others her fold the DNA -will create this little loops -the idea is to fold them close together so when the enzymes are responsible for making a cut, between last of exon and first of enzyme they hold it there so they don’t lose the first part of rna, bring closer to the next beginning of the exon, rid intron and have continuous coding sequences -you do this for every single intron -if have pre-mRNA for every typical aa protein, 27 to 30 000 nucleotides, must cut out introns and left with 12 to 1500 nucleotides in mature RNA, when translated, 3 nucleotides per amino acids, will end up with 4–600 aas -must happen to introns, and SNERPS are specific to them -goal is to grab both ends of the intron (base pairing), bring the exons closer, cutting the introns out, and putting
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