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Gene Expression in Eukaryotes

Gene Expression in Eukaryotes. mRNA Transcription and Processing. Eukaryotic Transcription Machinery. RNA polymerase II synthesizes all eukaryotic mRNA precursors composed of 12 different subunits remarkably conserved from yeast to mammals Polymerase II promoters

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Gene Expression in Eukaryotes

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  1. Gene Expression in Eukaryotes mRNA Transcription and Processing Karp/CELL & MOLECULAR BIOLOGY 3E

  2. Eukaryotic Transcription Machinery • RNA polymerase II • synthesizes all eukaryotic mRNA precursors • composed of 12 different subunits • remarkably conserved from yeast to mammals • Polymerase II promoters • 5' side of each transcription unit (mostly) • TATA box at 24-32 bases upstream from start site • Consensus: 5'-TATAAA-3' Karp/CELL & MOLECULAR BIOLOGY 3E

  3. Figure 11.18a Karp/CELL & MOLECULAR BIOLOGY 3E

  4. Eukaryotic Transcription Machinery • Initiation of transcription • Requires a number of general transcription factors (GTFs) • Their precise roles remain to be determined • General = conserved in a variety of genes and organisms • A preinitiation complex assembles at the TATA box • Required before Pol II binds • First: TATA-binding protein (TBP) • TBP has 10-stranded b sheet curved into a • saddle-shaped structure that sits astride the DNA • TBP is subunit of TFIID (fraction D) Karp/CELL & MOLECULAR BIOLOGY 3E

  5. Eukaryotic Transcription Machinery • TBP binding distorts DNA conformation • Bound DNA develops a distinct kink • DNA duplex becomes unwound over ~8 bp • TBP is a universal TF • mediates binding of all 3 eukaryotic RNA polymerases; • present in 3 different protein complexes • TFIID (pol II) • SL1 (pol I) • TFIIIB (pol III) Karp/CELL & MOLECULAR BIOLOGY 3E

  6. GTF’s and initiation • 3 GTFs interact with promoter on DNA • TBP of TFIID • TFIIA • TFIIB) • provide platform for multisubunit polymerase + TFIIF Karp/CELL & MOLECULAR BIOLOGY 3E

  7. GTF’s and initiation • followed by another pair of GTFs • TFIIE & TFIIH • TFIIH is only GTF known to possess enzyme activities • 2 of its subunits act as DNA helicases • allows polymerase access to template strand • Another TFIIH subunit functions as protein kinase • phosphorylate RNA polymerase Karp/CELL & MOLECULAR BIOLOGY 3E

  8. Figure 11.19 Karp/CELL & MOLECULAR BIOLOGY 3E

  9. Elongation • One GTF (TFIID) may be left behind at promoter • Future initiation? • Other GTF’s are released from the complex Karp/CELL & MOLECULAR BIOLOGY 3E

  10. (CTD) of largest polymerase II subunit • 7 amino acid sequence (-Tyr-Ser-Pro-Thr-Ser-Pro-Ser-); • in mammals, 52 repeats • All but 2 prolines are targets for phosphorylation • preinitiation pol II is nonphosphorylated • when transcribing, it is heavily phosphorylated • phosphorylation is likely a trigger for transcription • An elongation complex • a number of large accessory proteins • > 50 components • total molecular mass of >3 million daltons • Probably, template moves through immobilized machine Karp/CELL & MOLECULAR BIOLOGY 3E

  11. Figure 11.20 Karp/CELL & MOLECULAR BIOLOGY 3E

  12. Regulation • Specific transcription factors • bind to other sequences (CAAT-box, GC-box, enhancers) • activate (or prevent) preinitiation complex formation • Determine polymerase initiation rate Karp/CELL & MOLECULAR BIOLOGY 3E

  13. mRNA properties • They are found in the cytoplasm • They are attached to ribosomes when they are translated • significant noncoding, nontranslated segments • ~25% of each globin mRNA is noncoding • Noncoding portions are found on both 5' & 3' ends • have sequences with important regulatory roles • altered ends not seen in prokaryotes • 5' end - methylated guanosine cap • 3' end - string of 50 - 250 adenosine residues [the poly(A) tail] • histones are an exception Karp/CELL & MOLECULAR BIOLOGY 3E

  14. Figure 11.21 Karp/CELL & MOLECULAR BIOLOGY 3E

  15. Figure 11.22c Karp/CELL & MOLECULAR BIOLOGY 3E

  16. Split Genes • Philip Sharp et. al. (MIT) • Richard Roberts, Louise Chow et. al. (Cold Spring Harbor, NY) • Adenovirus introns • Alec Jeffreys & Richard Flavell (1977, U. of Amsterdam) • Exons - parts of gene that contribute to mature RNA product • b-globin gene • Introns - intervening sequences Karp/CELL & MOLECULAR BIOLOGY 3E

  17. Figure 11.23 Karp/CELL & MOLECULAR BIOLOGY 3E

  18. Figure 11.24 Karp/CELL & MOLECULAR BIOLOGY 3E

  19. Split Genes • Split genes found in simpler eukaryotes (yeast, protists) • fewer in number & smaller in size than in plants & animals • Introns are found in all types of genes (tRNAs, rRNAs, mRNAs) • Must be removed from primary transcript to make mature mRNA • Shirley Tilghman, Philip Leder, et al. (NIH) – • R-loop formation seen in EM • determined relationship between 15S & 10S (mature) globin RNAs Karp/CELL & MOLECULAR BIOLOGY 3E

  20. Figure 11.25a & b Karp/CELL & MOLECULAR BIOLOGY 3E

  21. Split Genes • Ovalbumin (protein found in hen's eggs) gene • 7 loops form; correspond to 7 introns • ~3 times more sequence than 8 exons • Individual exons in all genes are typically < 300 bases • Individual introns typically between 1,000 & 100,000 bases • Explains hnRNA length • Type I collagen gene • >20 times the length of mature message • contains >50 introns Karp/CELL & MOLECULAR BIOLOGY 3E

  22. Processing • Ribonucleoproteins convert transcript to mature mRNA • Addition of a 5' cap & a 3' poly(A) tail • Removal of any intervening introns • 5' methylguanosine cap forms very soon after RNA synthesis begins • 5' end initially has triphosphate • First enzyme produces diphosphate • Then, GMP is added in inverted orientation • 5'-5' triphosphate bridge • Next, guanosine base is methylated at position 7 • ribose methylated at 2' position • 5' end modifications occur very quickly Karp/CELL & MOLECULAR BIOLOGY 3E

  23. Processing • May serve several functions: • prevents exonuclease digestion of mRNA 5' end • aids in transport of mRNA out of nucleus • important in initiation of mRNA translation Karp/CELL & MOLECULAR BIOLOGY 3E

  24. The poly(A) tail • ~15 bases downstream from AAUAAA • a protein complex carries out processing at 3' end • associated with the RNA polymerase • Included is an endonuclease • poly(A) polymerase adds ~250 adenosines • protects the mRNA from premature degradation • Poly(A) tail allows affinity chromatography purification Karp/CELL & MOLECULAR BIOLOGY 3E

  25. Figure 11.28 Karp/CELL & MOLECULAR BIOLOGY 3E

  26. Figure 11.29 Karp/CELL & MOLECULAR BIOLOGY 3E

  27. Splicing • Must be absolutely precise • single base error changes reading frame • conserved sequence found at exon-intron junctions • Usually G/GU at 5' intron end (5' splice site) • Usually AG/G at 3' end (3' splice site) • ~1% of introns have AT & AC, respectively • processed by different spliceosome (U12 spliceosome) Karp/CELL & MOLECULAR BIOLOGY 3E

  28. Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.30

  29. Splicing • functional differences as yet undetected) • has U12 snRNA instead of the U2 snRNA of the major spliceosome • U12 spliceosomes • absent from yeast & nematodes • present in plants, insects & vertebrates • regions adjacent to intron contain preferred nucleotides • play big role in splice site recognition (exonic enhancers) • mutation can block intron excision Karp/CELL & MOLECULAR BIOLOGY 3E

  30. Splicing • human thalassemia caused by mutations in globin splice sites • RNA catalytic abilities led to understanding of splicing mechanism • Thomas Cech et al. (1982, U. of Colorado) • RNA catalysis in pre-rRNA • ciliated protozoan Tetrahymena (ribozymes) Karp/CELL & MOLECULAR BIOLOGY 3E

  31. Two types of intron splicing mechanisms described • Group I introns (Tetrahymena pre-rRNA) • most common in fungal/plant mitochondria, plant chloroplasts, & in nuclear RNA of lower eukaryotes, like Tetrahymena • variable sequence, but similar 3D structures Karp/CELL & MOLECULAR BIOLOGY 3E

  32. Two types of splicing • Group II introns • also self-splicing • seen in fungal mitochondria & plant chloroplasts • structure very complex & different from Group I introns • go through intermediate stage (lariat, like cowboy rope) • First step is cleavage of 5' splice site • followed by formation of lariat • covalent bond between intron 5' end & A near 3' end • 3' splice site cleavage releases lariat • allows exon cut ends to be ligated Karp/CELL & MOLECULAR BIOLOGY 3E

  33. Figure 11.32 Karp/CELL & MOLECULAR BIOLOGY 3E

  34. Two types of splicing • Animal pre-mRNAs are processed like Group II introns • difference is that intron cannot splice itself • needs snRNAs & their associated proteins Karp/CELL & MOLECULAR BIOLOGY 3E

  35. hnRNA’s • hnRNP’s facilitate processing reactions • spliceosomes remove introns • have a variety of proteins & snRNPs • assembled they bind to the pre-mRNA • snRNPs help remove introns from transcript Karp/CELL & MOLECULAR BIOLOGY 3E

  36. hnRNP’s • snRNPs required: • U1 snRNP, U2 snRNP, U5 snRNP & U4/U6 snRNP (U4 & U6 snRNAs bound together) • U6 is most likely to act as a ribozyme • makes both cuts in the pre-mRNA required for intron removal Karp/CELL & MOLECULAR BIOLOGY 3E

  37. Figure 11.35 Karp/CELL & MOLECULAR BIOLOGY 3E

  38. Figure 11.36 Karp/CELL & MOLECULAR BIOLOGY 3E

  39. snRNPs: a dozen or more proteins • One family, the Sm proteins are present in all of the snRNPs • they bind to one another & to a conserved site on each snRNA • forms the core of the snRNP • Sm are targets of autoimmune antibodies • systemic lupus erythematosus Karp/CELL & MOLECULAR BIOLOGY 3E

  40. snRNP’s • The other proteins of the snRNPs are unique to each particle • ATP-consuming, RNA helicases (unwind double-stranded RNAs) • helicases are found within snRNPs • at least 8 implicated in the splicing of yeast pre-mRNAs • snRNAs are catalytically active (not the proteins) • similar to group II introns, which splice themselves • snRNAs closely resemble parts of the group II introns Karp/CELL & MOLECULAR BIOLOGY 3E

  41. snRNP’s • The proteins likely serve supplementary roles • Maintaining the proper 3D structure of the snRNA • Driving changes in snRNA conformation • Transporting spliced mRNAs to the nuclear envelope • Selecting splice sites to be used Karp/CELL & MOLECULAR BIOLOGY 3E

  42. snRNP’s • snRNP proteins - not alone in mRNA processing • SR proteins: large number of SR dipeptides • thought to form interlacing networks that span intron/exon borders • They help recruit snRNPs to the splice sites • SR proteins have positive charge • may also bind electrostatically to negatively charged phosphate • assembly of splicing machinery occurs during RNA synthesis • most of RNA processing machinery travels with polymerase Karp/CELL & MOLECULAR BIOLOGY 3E

  43. Figure 11.37 Karp/CELL & MOLECULAR BIOLOGY 3E

  44. snRNP’s • Most genes contain a number of introns • splicing reactions occur repeatedly on single 1° transcript • introns may be removed in preferred order • generates specific processing intermediates whose size lies between Karp/CELL & MOLECULAR BIOLOGY 3E

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