SectionNotesweek9 - Section Notes week 9 GSI: Nikki...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Section Notes week 9 GSI: Nikki R. Kong Eukaryotic transcription regulation Many different general types of mechanisms: 1. Protein  ­> mRNA: txn output is regulated by the amount of transcription factors around, which determines the amount of mRNA made 2. Ligand binds protein  ­> mRNA: similar to cAMP binding CAP protein in bacteria Lac operon regulation; also glucocoticoid receptor in the nuclear receptor family needs ligand binding to enter the nucleus 3. Small modifications such as phosphorylation: many transcription factors that have transactivation domains are often modified by kinases or other enzymes. These modifications are then recognized by co ­factors, examples given in lecture was CREB protein whose phosphorylated form is recognized by a HAT protein complex, CBP/P300 4. Protein ­protein interactions: a second protein is required to stimulate transcription activity, such as dimerization, or a second protein that has no DNA ­binding ability of its own, but stimulates the already ­bound transcription factor. 5. Protein/inhibitor where the inhibitor has to be removed (sometimes by phosphorylation) for the transcription factor to be active; similar to 6, but this can take place in the nucleus 6. Protein/inhibitor by sequestration: an inhibitor might latch on to the transcription factor or shields its nuclear localization signal (NLS). Only once the inhibitor is removed by either degradation or another mechanism, can the transcription factor localize to the nucleus and activate transcription. Example given in class was NF ­kappaB 7. Receptor in the plasma membrane, and following signal, part of the receptor becomes separated and translocates into the nucleus to turn on transcription. Such as SREBP or Notch receptors, the latter undergoes a proteolytic cleavage to release the intracellular domain of Notch, which goes into the nucleus to act as a co ­activator of transcription. CREB: cAMP response element binding protein  ­ ­> Responds to growth hormones or neurotransmittors depending on the cell type  ­ ­> Only genes containing CRE sequences in their promoters can be activated through this pathway  ­ ­> b ­zip transcription factor factor that is regulated by ade involving cAMP response REB = leucine zipper trxn factor FYI only) that is only active when phosphorylated of a transcription factor f ple ytosolic inhibitor protein athat is regulated by a c Cyclic AMP-inducible gene expression ctivates R=regulatory subunit; C ­ catalytic (i.e. kinase) rotein kinase trc in that f nuclear factor ­ Nuclear NA translocatesit only binds DNA The free catalytic subunit transcriptionentry. ­>ofSpimilar to entry is stimulated by when phosphorylated, but different since it still can’t by itself recruit Pol II. to the (directly phosphorylates the ranscription immunethat nucleus and stimulates ~150 gtenes), is activated bysignal on NFκB. ystem tein sactor CREBasks t ­he Pnuclearcells in response to m are released bhosphorylated CREB  ­> y nearby localization f kin 1 (IL-1), which(CRE-binding protein), leading to expression recruits CBP/P300 HAT and subsequently the basal transcription machinery , NF-!B of cAMP-inducible genes. by other stressful situations, such can also be activated NF ­kappaB: responds to inflammation and ionization, or infection  ­ ­> NF ­kappaB has two subunits: p65 and p60, and there’s a nuclear localization signal (NLS) on both that’s shielded by bound I ­kappB in the cytoplasm. I ­kappaB needs to phosphorylated, ubiquitinated, and then degraded by the proteasome for NF ­kB to translocate into the nucleus. Ubiquitin chains attached to proteins can direct them to the proteasome for degradation Ubiquitin is a small protein that can be attached to other proteins to tag them for degradation or other cellular fates. Three enzyme are involved in putting ubiquitin chains onto a target protein. E1 = ubiquitin activating enzyme E2 = ubiquitin conjugating enzyme E3 = ubiquitin ligase - directs transfer or Ub from E2 onto the target. E3 is specific for the target protein and binds the target as well as the E2. The proteasome is a molecule that degrades proteins that have been targeted for degradation by Ubiquitination. It binds the ubiquitin chain and then unfolds the protein and feeds it in to the core chamber where it is chewed up into its amino acid precursors and spit back into solution. the phosphorylated form of IκBα is targeted for . degradation by ubiquitination by E3 ligase. lid and base core particle regulatory particle now that IκBα is gone the nuclear localization signals are exposed which will cause the protein to be transported into the nucleus where it can stimulate transcription Mani A , Gelmann E P JCO 2005;23:4776-4789 ©2005 by American Society of Clinical Oncology hormone, a DNA-binding domain, and a region that I K types of transcription W D 50 T S activates transcription of the regulated gene. The highly S N factors, but don’t need conserved DNA-binding domain has two zinc fingers. C C C 10 C to know that GR is a The sequence shown here is that for the estrogen Q Q 60 receptor, but the residues in bold type are common to N E V zinc finger, but should Zn T Zn all steroid hormone receptors. A A know the three G A P domains below C C C C Nuclear receptors: example is glucocorticoid receptor, 2 Zn fingers in D domain Lehninger. Biochemistry. 4th ed 30 40 70 80 MKETRY KAFFKRSIQGHNDYM RLRKCYEVGMMKGGIRKDRRGG n ­Variable txn activation domain (A)—DNA binding (D)—ligand binding (L) ­c H 3N COO Transcription activation (variable sequence and length) DNA binding (66–68 residues, highly conserved) Hormone binding (variable sequence and length) Model of hormone-dependent gene activation by the glucocorticoid receptor (GR)  ­ ­> Ligands are usually lipids, thus they can cross plasma membrane to find the receptors in the cytoplasm  ­ ­> Usually the L domain is bound by HSP90 type of inhibitors, ligands compete away inhibitors and stimulate nuclear entry of the receptors Immunofluorescence (e.g.Hsp90) Immunofluorescence It’s good for visualizing where a protein is localized in the cell, its relative abundance, Ex: Glucocorticoid and whether it colocalizes with something else (which also needs to be fluorescently receptor transcription detected some how). factor Confocal immunofluorescent analy - labeled different proteins with sp each with a different color fluorop a) b) Gau This is the best diagram I could find. Were where it says antigen, that would be whatever protein you’re interested in visualizing. To visualize your protein of interest you need a specific antibody against it and then need to label the antibody with a fluorophore. They were looking at two different forms (b) and where they mutated the nuclear protein is stained with a red fluorophore for the protein. The blue is staining of th the protein is nuclear and when it’s cyto h Confocal immunofluorescent analysis of mouse testis - labeled different proteins with specific antibodies each with a different color fluorophore attached a) b) Gau X et al. 2008. JBC ould be whatever est you need a uorophore. They were looking at two different forms of a protein - wild type (b) and where they mutated the nuclear export signal (a). The protein is stained with a red fluorophore conjugated to an atibody for the protein. The blue is staining of the nucleus to show when the protein is nuclear and when it’s cytosolic. Processing mRNA  ­ ­> Capping, poly(A) tail, and splicing are all coupled with Pol II and transcription for efficiency  ­ ­> Structural organization of a pre ­mRNA 5’cap ­5’UTR ­ ­ ­EXON1 ­ ­ ­ ­ ­intron1 ­ ­ ­ ­ ­EXON2 ­ ­ ­ ­ ­intron2 ­ ­ ­ ­ ­EXON3—3’UTR ­AAAAA.. Capping Protects mRNA from 5’ ­3’ exonucleases, facilitates splicing, export, and translation in eukaryotes (recognized by the ribosome) Features: single nucleotide guanine cap that is N7 ­methylated with a 5’ ­5’ linkage to the beginning of the mRNA  ­ ­> The cap is added after ~25nt of the mRNA is made and Pol II is paused (because of NELF and DSIF)  ­ ­> Two enzymes in the same complex are needed: 1. Phosphohydrolase, 2. Guanylyltransferase which is recruited by phosphorylated Ser5 on the Pol II CTD.  ­ ­> Methylation of G comes after it’s attached, by a methyltransferase with S ­ adenosylmethionine as a methyl donor Poly(A) tail Protects mRNA from 3’ ­5’ exonucleases, also facilitates translation and export. Almost all eukaryotic mRNAs have poly(A) tails except for histones, which are transcribed dependent on cell cycle (during early S phase) and form hairpin structures at the end.  ­ ­> Specific sequences direct where the poly(A) tail is added: AAUAAA followed by a G/U ­rich region with the cleavage/Poly(A) addition site in between, about 10~35nt after the AAUAAA site.  ­ ­> A cleavage/recognition factor (CPSF) and its stimulatory factor (CstF) bind to the AAUAAA sequence, and only cleaves after poly(A) polymerase (PAP) is also recruited! This is important because the 3’OH generated from the cleavage is very reactive, and thus must be immediately extended by AAA addition.  ­ ­> Initial polyadenylation by PAP is slow, but after 12As have been added, a protein called polyA binding protein II (PABPII) binds to the short (A) tail and accelerates polyadenylation by PAP until the tail is 200~250nt in eukaryotes.  ­ ­> Alternative poly(A) signal (AAUAAA) can control where and what the 3’UTR sequence is. 3’UTR is often target of regulatory small RNAs called microRNAs. Since binding of microRNAs to mRNA often leads to destruction of the mRNA molecule, alternative poly(A) signals can contribute to mRNA stability as well. Splicing The mRNA made so far is called a pre ­mRNA which contains both introns and exons There are self ­splicing introns in some pre ­mRNA in both bacteria and eukaryotes: Group I: uses an outside guanine as a nucleophile for transesterification reaction Group II: uses an internal A residue as a nucleophile for splicing In eukaryotes, most mRNAs are spliced by the spliceosome (as covered in class) Spliceosome {non ­RNP proteins such as helicases, + U 1, 2, 4, 5, 6 RNPs (ribonucleotide protein, both protein and RNA) [each containing an snRNA (U 1, 2, 4, 5, 6) + 6~10 other proteins]} All vertebrates have introns that start with GU (5’ splice site) and end with AG (3’ splice site), most have an AG ­rich region after GU, and a pyramiding ­rich region (YYYY) before AG, and a 100% conserved A branch point 5’ of YYYY region 1. Recognition: needs ATP  ­ ­> snRNA in U1 RNP base pairs with AG, and snRNA in U2 RNP base pairs with A branch pt region. 2. Before recognition, U4 masks U6; but after recognition, rearrangements happen and U6 and U2 form a catalytic center. Note: catalysis is carried out by RNAs, not proteins, and these RNA molecules are called ribozymes. 3. Splicing—2 transesterification reactions: no ATP required  ­ ­> “trans” because the total number of ester bonds are the same. i. U2/U6 catalytic center activates the branch pt A as a nucleophile which attacks the bolded phosphate at the 5’ splice site: Exon1 ­pGpUp ­ ­ ii. above reaction generates a lariat structure with the branch pt A as the “knot” 8885d_c26_995-1035 2/12/04 11:18 AM Page 1012 mac34 mac34: kec_420: iii. Finally Exon1 ­3’OH generated from the first reaction attacks the bolded phosphate at the 3’ splice site: lariat ­ ­ApGp Exon2 RNA in snRNP proteins recognizes sequence The central region of the intron, which mayU2 range from 40 bases to 500 kilobases in U1 length, generally is unnecessary for splicing to occur. 5 3 UCCA 5 Exon CAUA AUGAUGU AGGUAGGU UACUA C A AGGU 3 Exon (a) sequence that If you mutation in the the pairs with snRNPs and it If you have ahave a mutation in sequence that base pairs with snRNPs and destroys destroys could you find out yout’s becauseit f base pairing and not because it splicing, how could if i find out if ois because of base pairing splicing, how anRNP because n disrupts snRNP 3binding in some other way? nd 5 disrupts sG U notbinding iit A ome G ther way? s Ao Spliceosome Make compensatory mutation. U1 snRNP Compensatory mutations: mutations in the binding partner that should Compensatory mutation = mutation in the binding account for the ATP of base pairing. For example, if you had a G ­>A mutation in loss CTD U2 snRNP partner that should complement the mutation. So if had a G to the mRNA, yADPcan itry to make a C ­>U mutation in the snRNA to see if it CBC ou P allows a A mutation in the mRNA you could try making a C to T Cap binding again.utation in the snRNP RNA to see if that allows it to bind m 5 GU U1 again3 U2 Alternative splicing ATP ADP AG A U4/U6 U5 Pi 1 in 20 mRNA molecules is alternatively spliced in eukaryotes to generate protein diversity and as an additional level of gene regulation Inactive U4/U6 spliceosome You can have alternative spliced isoforms making different proteins that perform A different 5unctions in distinct cell tG 3 f GU A ypes. U5 U2 U1 1 type of mechanism for alternative splicing involves SR proteins and their binding sites, ESE (exonic splicing enhancers), in exons to be retained ATP  ­ ­> SR proteins interact with each other and promote binding of U1 to the 5’ splice U1, U4 ADP Pi site of this otherwise weak exon that won’t normally have U1 bound to GU in its downstream intron. Then U2AF binds YYYY region and 3’ splice site, and recruits U2 Active snRNP to the branch pt U5. This forms a cross ­exon recognition complex that can A spliceosome retain this exon in the final mRNA A GU 5 3  ­ ­> SR proteins are regulated A s well; they are a family with different SR proteins aG U6 U2 favoring difference sites, also require additional proteins to help them bind ESEs. lariat Spliced intron formation For example: (c) Ua In drosophila fruit flies, there 5 re three proteins responsible for sex determination: primary transcripts. (a) RN U FIGURE 26–16 Splicing mechanism in mRNA G sex ­lethal (sxl), transformer (tra), and doublesex (dsx). interactions in the formation of spliceosome complexes. The U1 A pairing OH 1. tra is made differently in female and 3male flies due to alternative splicing, nd that is complementary to the splic snRNA has a sequence near its 5 e the AG 5 site at the ranslation stop codon, s of male fly gets an extra U6 U2 which contains a premature t5 end of the intron. Base pairing o U1 to this region of the exon 3 U5 primary transcript helps define the 5 splice site during spliceosome males make an inactive form of tra. U G A U6 U2 A G Intron release 5 3 (b) assembly ( is pseudouridine; see Fig. 26–24). U2 is paired to the intron a position encompassing the A residue (shaded pink) that becomes the nucleophile during the splicing reaction. Base pairing of U2 snRNA caus a bulge that displaces and helps to activate the adenylate, whose 2 OH will form the lariat structure through a 2 ,5 -phosphodiester bond. (b) Assembly of spliceosomes. The U1 and U2 snRNPs bind, then th 2. The female ­specific dsx gene requires exon 4 to be retained to be functional, and exon 4 of dsx contains ESE sequences 3. tra’s gene product is required to recruit SR proteins onto the ESE sites in dsx exon 4. Therefore, only in female flies with an active tra protein, can dsx exon 4 be retained to generate a functional dsx protein. ...
View Full Document

This document was uploaded on 09/12/2011.

Ask a homework question - tutors are online