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Lecture 6

Cell Biology - Lecture 6 -Video 3 - Notes

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CAS BI 203
Martin Steffen

Lesson 6 – Video 3 [00:00:00.00] 2580 [00:00:01.40] SPEAKER 1: To conclude our discussion of gene expression and transcription and 2581 translation, we have a few topics that will sort of round out our perspective on the entire process. 2582 That includes talking about the way that introns are spliced out of the initially synthesized 2583 eucaryotic mRNAs, the transport of mRNA from the nucleus to the cytoplasm. We'll talk about 2584 how protein folding can be assisted by chaperones. 2585 [00:00:32.09] We'll talk about how proteins are degraded. We'll talk a little bit about the 2586 proteasome which is the protein complex that degrades the cellular protein sort of the anti2587 partner the ribosome. And then we'll just talk a little bit about how antibacterial drugs target the 2588 ribosome as a way of trying to cause bacterial cell death. 2589 [00:01:02.03] Here I'd like to just briefly show some of the data that was used for the discovery 2590 of introns.As you can see in this case where you're going to hybridize some mRNAto DNAto 2591 find out where the gene is, if there are no introns, the mRNA sequence will be an exact copy of 2592 the genomic DNA. 2593 [00:01:25.78] However, if the mRNA have introns spliced out, part of the mRNAwill hybridize 2594 to part of the genomic DNAfor exon one. Genomic DNAcorresponding to the intron will not 2595 hybridize to anything in the mature mRNA, but then this other part of the mRNA will hybridize 2596 to part of the genomic DNAfor exon two.And these kinds of structures are observed like, for 2597 instance, here.And that was how the existence of introns was deduced. 2598 [00:02:06.53] Just like the sequences that help proteins recognize where transcription should 2599 start and stop, there are sequences that help other proteins and RNAmolecules recognize that 2600 these are exon/intron boundaries and that there should be a splicing event here. Now not all of 2601 the exons are used and not all splices are simple splices with the contiguous regions being 2602 spliced together. Sometimes you might skip exon two and go to exon three. The way that that is 2603 determined by the cell that is not entirely clear as far as I'm aware. 72 [00:02:52.33] And there is an eight base pair sequence at the 2604 5' end and 12 base pair sequence of 2605 the 3' end that indicates where the splicing should occur.And here is where the excised intron 2606 would have been. 2607 [00:03:15.67] Now in the sequences here, in addition toACGU, there's some other characters 2608 here, other letters. That's Y is for pyrimidine. R is for purine, meaning it could be either Aor G. 2609 Pyrimidine means it could be either U or C. 2610 [00:03:37.17] In the center here, you see some of the sequences that are involved, in particular, 2611 theAwhich does some of the chemistry of the splicing reaction which is also done by RNA 2612 molecules. 2613 [00:03:49.64] Splicing is performed by a protein and RNAcomplex called the spliceosome. It's a 2614 very large, complex machine.And an example of the part of the RNAdoing some of the 2615 chemistry involved with the splicing of exons is that base pairAthat was in red in the previous 2616 slide attacking the upstream exon intron boundary forming this laureate structure which then is 2617 cleaved our. The two exon ends are joined.And now you have a spliced boundary region of your 2618 mRNA. 2619 [00:04:39.43] Now this is one of the first examples of RNAhaving enzymatic activity. And the 2620 fact that RNAis a molecule that can both code for information and perform lots of biochemistry 2621 has led some people to speculate on the possibility of RNAbeing one of the very primordial 2622 molecules present at the origin of life.And that's as good a hypothesis as any right now. 2623 [00:05:11.73] One of the differences between eucaryotic and procaryotic RNAthat we've 2624 pointed out was a poly(A) tail at the three prime end of eucaryotic mRNAs. Once the RNAstop 2625 sequence for RNApolymerase has been passed, the RNApolymerase will dissociate from the 2626 DNA, dissociate the RNAfrom the complex, and an enzyme called poly(A) polymerase, a 2627 polymerase which does not need a template, will just start addingA,A,A,A,Ato the end of a 2628 synthesized mRNA. 2629 [00:06:07.08] This poly(A) run will be covered with poly(A) binding protein, and it will help be 2630 involved in the transport of mRNA out of the nucleus. The fact that all eucaryotic genes, mRNAs 2631 end in poly(A)-- those that are protein coding-- has been tremendously useful because it allows 2632 scientists to take a poly(T) primer which will bind to all the RNAs and synthesize them for 2633 making molecules called CDNA.And we'll see that in a future lecture. 2634 [00:06:47.89] Once synthesized and processed in the nucleus, mRNAs need to be transported out 2635 into the cytoplasm. In the nucleus, they're bound by the cap-binding protein, the poly(A) binding 2636 proteins, and a complex called the exon junction complex. Movement inside the nucleus is 2637 partially aided by diffusion once it goes through the nuclear pore large openings in the double 2638 membrane that separates the nucleus from the cytosol. Proteins will bind to the cap region and 2639 the mRNAs and help transport them to ribosomes at the sites where they are synthesized into or 2640 read for protein synthesis. 73 [00:07:42.09] We saw earlier in the course that proteins could be 2641 unfolded with urea full length 2642 protein sequences, could be unfolded with urea, and then refold on their own.As they're being 2643 synthesized, the amino end of a protein is exposed prior to completion of the entire polypeptide 2644 chain. So proteins can start to fold in domains before the entire sequence is even available. 2645 [00:08:13.63] This shows you that kinetic factors potentially can play an important role in the 2646 final folded structure. If this portion of the protein starts to lock into a particular configuration, 2647 the once full length protein is present, it may not be able to unfold what's already been 2648 performed. And this is one of the reasons why we're not certain that most proteins fold to a 2649 global thermodynamic minimum because these proteins can get kinetically trapped into a local 2650 minimum by the process where part of the protein starts to fold before the rest is present. 2651 [00:09:02.66] Sometimes proteins don't get folded correctly due to ra
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