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Nucleus PowerPoint Presentation


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  1. Gene Expression: Multiple, Spatially and Temporally Distinct Steps Carried out by Distinct Cellular Machinery Nucleus DNA Pol II transcription Primary RNA transcript Nuclear processing Capping Splicing Polyadenylation Degradation Cytoplasm Mature mRNA Mature mRNA Protein Export Translation - Regulation Can Be at Several Different Levels - Dynamic Protein Association

  2. How can the cell distinguish between intron-containing pre-mRNAs (2) spliced mRNAs (3) intronless mRNAs to ensure that (1) are retained in the nucleus while (2 and 3) are exported to the cytoplasm?

  3. Mechanisms of mRNA turnover (NMD, mRNAs contain a premature stop codon, an in-frame stop codon within a retained intron, or an extended 3’ UTR due to improper polyadenylation site usage) (mRNAs lack an in-frame termination codon-decay occurs in the cytoplasm)

  4. Nuclear retention of unspliced mRNAs • An intact 5’ splice site and branchpoint are • required for nuclear retention of pre-mRNAs • Numerous splicing factors, including U1 • snRNA and branchpoint binding protein • (BBP/SF1), have been found to affect nuclear • retention of pre-mRNAs • In yeast, perinuclearly located Mlp1 physically • retains improperly spliced pre-mRNAs but • does not affect the splicing process itself • Thus, it appears that Mlp1 retains pre-mRNAs • that assemble into a spliceosome but fail to • proceed through splicing before reaching the • nuclear pore complex green-Mlp1 detected with an antibody red-the nucleolar protein Nop1 detected with an antibody Galy, V. et al. Cell 116, 63-73 (2004)

  5. Coupling of Transcriptional and Post-Transcriptional EventsmRNA Surveillance RNA-Mediated Gene Silencing

  6. Coupling of Transcriptional and Post-Transcriptional Events A) CTD of RNA Pol II - binding platform for mRNA processing components B) TREX - couples TRanscription and EXportC) Exon Junction Complex (EJC) - splicing mark and coupler

  7. mRNA Surveillance Quality Control Mechanism - ‘Process vs. Discard’ B) Nonsense Mediated Decay - Elimination of mRNAs with Premature Stop Codons (PTCs)

  8. RNA-Mediated Gene Silencing Post-Transcriptional Gene Silencing (PTGS or RNA Interference) - mRNA degradation - translation block B) Transcriptional Gene Silencing (TGS) - DNA methylation - Heterochromatin formation - DNA rearrangement/elimination

  9. Gene Expression: Linear Assembly Line? Nucleus DNA Pol II transcription Primary RNA transcript Nuclear processing Capping Splicing Polyadenylation Degradation Cytoplasm Mature mRNA Mature mRNA Protein Export Translation

  10. Gene Expression: Complex Network of Coupled Interactions

  11. Co-Transcriptional Recruitment of pre-mRNA Processing Factors TREX 5’ Nascent mRNA CTD • 5’ Capping Enzymes • Splicing Machinery • 3’ Cleavage/PolyA Factors • Export Receptors

  12. T-REX Transcription Export Transcription Elongation Factors (THO Complex: Hpr1p, Tho2p, Mft1p, Thp2p) Export Factors (Yra, Sub2) Tex1 (unknown function)

  13. Nuclear mRNA Surveillance and Quality Control: Process or Discard Degradation by Nuclear Exosome (3’-5’ exonucleases)

  14. TranscriptionalCoupling & mRNA Surveillance Nascent mRNA RNA Pol II NPC Ribosome Exosome

  15. Exon Junction ComplexThe Splicing Process Leaves its ‘Mark’ • The EJC assembles following splicing. • EJC is deposited 20-24 nts upstream of the exon-exon junction. • EJC as a ‘Molecular Link’ between splicing and downstream events. EJC

  16. EJC: A Molecular Link Between mRNA Splicing and Subsequent Events (Export, Localization, Decay, Translation, etc.)

  17. Common Trigger: RNA-Mediated Gene Silencing RNA Interference (PTGS) Transcriptional Gene Silencing (TGS)

  18. DICER Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs dsRNA processing ~22nt siRNAs recognition RISC (Argonaute) target mRNA degradation RISC: RNA-Induced Silencing Complex (contains effector nuclease)

  19. MicroRNAs and SiRNAs: Small but Mighty ‘Riboregulators’! - Viruses - Transposons - Repeat Elements - Exogenous Endogenous Host genes MicroRNA Precursor mRNA Degradation Translational Repression

  20. Proposed Biologic Roles ‘Immune System’ of the Genome • Antiviral Defense • Suppress Transposon Activity • Gene Regulation (Silencing) • (e.g. MicroRNAs, Heterochromatin)

  21. RNA-Mediated Gene Silencing RITS RNA-Induced Initiator of Transcriptional Gene Silencing (siRNAs, Ago1, Chp1 (chromodomain protein), Tas3) RITS RITS DNA methylation - Multiple dsRNA Inputs - Different Silencing Events

  22. Mechanism of RNAi-Mediated Heterochromatin Formation Trigger: Repetitive DNA RITS DNA 1. Histone 3 Methylation 2. Recruitment Two Very Different Outcomes!


  24. Molecular Biology Familiarity with basic concepts is assumed, including: • nature of the genetic code • maintenance of genes through DNA replication • transcription of information from DNA to mRNA • translation of mRNA into protein.

  25. Genetic code The genetic code is based on the sequence of bases along a nucleic acid. Each codon, a sequence of 3 bases in mRNA, codes for a particular amino acid, or for chain termination. Some amino acids are specified by 2 or more codons. Synonyms (multiple codons for the same amino acid) in most cases differ only in the 3rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized.

  26. Prokaryotic genes Prokaryotes (intronless protein coding genes) Upstream (5’) Gene region promoter Downstream (3’) TAC DNA Transcription (gene is encoded on minus strand .. And the reverse complement is read into mRNA) ATG mRNA 5´ UTR 3´ UTR CoDing Sequence (CDS) ATG Translation: tRNA read off each codons, 3 bases at a time, starting at start codon until it reaches a STOP codon. protein

  27. Prokaryotic genes (operons) Prokaryotes (operon structure) upstream promoter downstream Gene 1 Gene 2 Gene 3 In prokaryotes, sometimes genes that are part of the same operational pathway are grouped together under a single promoter. They then produce a pre-mRNA which eventually produces 3 separates mRNA´s.

  28. Bacterial Gene Structure of signals • - translation binding site (shine-dalgarno) 10 bp upstream of AUG (AGGAGG) • - One or more Open Reading Frame • start-codon (unless sequence is partial) • until next in-frame stop codon on that strand .. • Separated by intercistronic sequences. • - Termination

  29. Eukaryotic Central Dogma In Eukaryotes ( cells where the DNA is sequestered in a separate nucleus) The DNA does not contain a duplicate of the coding gene, rather exons must be spliced. ( many eukaryotes genes contain no introns! .. Particularly true in ´lower´ organisms) mRNA – (messenger RNA) Contains the assembled copy of the gene. The mRNA acts as a messenger to carry the information stored in the DNA in the nucleus to the cytoplasm where the ribosomes can make it into protein.

  30. tRNA The genetic code is read during translation via adapter molecules, tRNAs, that have 3-base anticodons complementary to codons in mRNA. "Wobble" during reading of the mRNA allows some tRNAs to read multiple codons that differ only in the 3rd base. There are 61 codons specifying 20 amino acids. Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for chain initiation. Mammalian cells produce more than 150 tRNAs.

  31. tRNA ( transfer RNA) is a small RNA that has a very specific secondary and tertiary structure such that it can bind an amino acid at one end, and mRNA at the other end. It acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mRNA. T Three-dimensional Tertiary structure Secondary structure of tRNA

  32. RNAstructure: MostRNAs have secondary structure, consisting of stem & loop domains. Double helical stemsarise from base pairing between complementary stretches of bases within the same strand. Loops occur where lack of complementarity, or the presence of modified bases, prevents base pairing.

  33. The “cloverleaf” model of tRNA secondary structure emphasizes the 2 major types of secondary structure, stem and loop domains. tRNAs typically include many modified bases, particularly in the loop domains. Tertiary structure depends on interactions of bases at more distant sites. Many of these interactions involve non-standard base pairing and/or interactions involving three or more bases. tRNAs usually fold into an L-shaped tertiary structure.

  34. Extending out from the "acceptor stem", the 3' end of every tRNA has the sequence CCA. The appropriate amino acid is attached to the ribose of the terminal A (in red) at the 3' end. The anticodon loop is at the opposite end of the L shape.

  35. Tertiary base pairs Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA. This example is from NDB file 1TN2. H atoms are not shown.

  36. Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA. The reaction occurs in two steps. In step 1, an O atom of the amino acid a-carboxyl attacks the P atom of the initial phosphate of ATP.

  37. In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.

  38. Aminoacyl-tRNA Synthetase Summary of the 2-step reaction: 1. amino acid+ ATPaminoacyl-AMP + PPi 2. aminoacyl-AMP+tRNAaminoacyl-tRNA+ AMP The 2-step reaction is spontaneous overall, because the concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

  39. There is a different Aminoacyl-tRNA Synthetase (aaRS) for each amino acid. Each aaRS recognizes its particular amino acid and the tRNAs coding for that amino acid. Accurate translation of the genetic code depends on attachment of each amino acid to an appropriate tRNA. Domains of tRNA recognized by an aaRS are called identity elements. Most identity elements are in the acceptor stem & anticodon loop. Aminoacyl-tRNA Synthetases arose early in evolution. The earliest aaRSs probably recognized tRNAsonly by their acceptor stems.

  40. Two different ancestral proteins evolved into the 2 classes of aaRS enzymes, which differ in the architecture of their active site domains. They bind to opposite sides of the tRNA acceptor stem, resulting in aminoacylation of a different OH of the tRNA (2' or 3'). There are 2 families of Aminoacyl-tRNA Synthetases: Class I & Class II.

  41. Class I aaRSs: Identity elements usually include residues of the anticodon loop & acceptor stem. Class I aaRSs aminoacylate the 2'-OH of adenosine at their 3' end. Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. Class II aaRSs tend to aminoacylate the 3'-OH of adenosine at their 3' end.

  42. Proofreading/quality control: Some Aminoacyl-tRNA Synthetases are known to have separate catalytic sites that release by hydrolysis inappropriate amino acids that are misacylated or mis-transferred to tRNA. E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a small percentage of the time activates the closely related amino acid valine to valine-AMP. After valine is transferred to tRNAIle, to form Val-tRNAIle, it is removed by hydrolysis at a separate active site of IleRS that accommodates Val but not the larger Ile. In some bacteria, editing of some misacylated tRNAs is carried out by separate proteins that may be evolutionary precursors to editing domains of aa-tRNA Synthetases.

  43. Some amino acids are modified after being linked to tRNA. • E.g., in prokaryotes & in mitochondria the initiator tRNAfMet is first charged with methionine. Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine moiety, using THF as formyl donor, to yield fMet-tRNAfMet. • In some prokaryotes, a non-discriminating aaRS loads aspartate onto tRNAAsn. The aspartate moiety is then converted by an amido-transferase to asparagine, yielding Asn-tRNAAsn. Glu-tRNAGln is similarly formed and converted to Gln-tRNAGln in such organisms.


  45. Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes.Mitochondrial and chloroplast ribosomes differ from both examples shown. Ribosome Composition (S = sedimentation coefficient)

  46. Structures of large & small subunits of bacterial & eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction. Consistent with predicted base pairing, X-ray crystal structures indicate that ribosomal RNAs (rRNAs) have extensive secondary structure.