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Five Classes of Introns Archaeal introns (tRNAs and rRNAs)

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Presentation on theme: "Five Classes of Introns Archaeal introns (tRNAs and rRNAs)"— Presentation transcript:

1 Five Classes of Introns Archaeal introns (tRNAs and rRNAs)

2 Generic Splicing Reaction Intron Two Steps ( “ Scissors than Tape ” ): Step 1: Break phosphodiester bonds at the exon-intron boundaries (splice junctions). 5 ’ bond broken before 3 ’ bond Step 2: Formation of a new phosphodiester bond between 3 ’ end of upstream exon and 5 ’ end of downstream exon Exon 2Exon 1 Exon 2 5 ’ splice junction3 ’ splice junction Intron

3 Splicing of Group I, II, and Pre-mRNA introns results from two sequential transesterification reactions “ Transesterification ” occurs when a hydroxyl group makes a nucleophilic attack on a phosphodiester bond to form a new phosphodiester bond while displacing a hydroxyl group The reaction requires no energy (ATP-independent) Phosphate is conserved Transesterification

4 Group I Introns Location: Nuclear rRNA genes of unicellular organisms (e.g. tetrahymena + other ciliates) Organellar tRNAs and rRNAs (mitochondria and chloroplasts) rRNA, mRNA, tRNA in bacteria (but rare) Viruses (e.g. T4 thymidylate synthase mRNA gene) Not found in vertebrates (e.g. “ us ” ) Role as Mobile Genetic Elements: introns can encode homing endonucleases that allow intron mobility

5 Group I Intron Structure Little conservation of primary structure (e.g. P, Q, R, S elements, 3 ’ splice-site G) All group I introns fold into a characteristic secondary structure (and likely tertiary structure) X-ray structure has been solved for most of the intron from tetrahymena rRNA RNA folding is critical for splicing

6 Group I Secondary Structure Internal Guide Sequence (IGS) G Binding Site (Active Site) Conserved G

7 Group I Intron Splicing Mechanism Autocatalytic or “ Self-splicing ” Sequential Transesterfications: Step I: 3 ’ OH of an exogenous guanosine attacks the phosphodiester bond at the 5 ’ splice site -G covalently linked to intron -5 ’ exon now contains a 3 ’ OH group Step II: 3 ’ OH of 5 ’ exon attacks the phosphodiester bond of 3 ’ splice site -intron is released -exons are ligated together Step 1 Step 2 G OH G G G intron Joined exons (mature RNA) 3’ exon 5’ exon intron 3’ exon5’ exon

8 Group II Introns Location: rRNA, tRNA, mRNA Eukaryotic organelles -mitochondria (fungi), chloroplasts (plants) mRNA of some Eubacteria (i.e. prokaryotes) Splicing: Autocatalytic or self-splicing in vitro proteins required in vivo Role as Mobile Genetic Elements: Introns often encode reverse transcriptases that allow intron to change genomic position.

9 Structure of Group II Introns Group II introns exhibit little primary sequence conservation All fold into a common secondary structure containing six helical domains (d1-d6) that emanate from a “ central wheel ” Domains 5 and 6 contain important catalytic activity

10 Tertiary Interactions Critical for Splicing of Group II Introns Exon binding sequences (EBS 1 and 2) in domain I to intron binding sequences (IBS 1 and 2) near 5 ’ end of 5 ’ exon (helps define 5 ’ splice-site) Nucleotides in loop of domain 5 interact with nucleotides in domain I Nucleotides in “ wheel ” (RGA=  ) interact with 3 ’ splice site (YA=  ’ ) (helps define 3 ’ splice-site) Nucleotides in in domain 1(  ’ ) interact with those near 5 ’ splice site (  )

11 Group II Introns 5 ’ EXON 3 ’ EXON “ Catalytic Core ” (Active Site) Branch Point Adenosine

12 Splicing Mechanism for Group II and Pre-mRNA Introns 2 ’ to 5 ’ Linkage 3 ’ to 5 ’ Linkage Lariat Intermediate Phosphate is conserved

13 Nuclear Pre-mRNA Introns Location: Common in vertebrates, numerous introns/gene Rare in unicellular eukaryotes like yeast, usually one intron/gene when any Conserved Sequences: at splice junctions (GT-AG rule), branch site and polypyrimidine tracts 5 ’ splice sitebranch site polypyrimidine tract 3 ’ splice site yeastAG/GUAUGUUACUAACYnCAG/G metazoans:AG/GURAGUYNCURACYYYYnYAG/G A in branch site adenosine is called the branch point Spacing between the elements is important The 5 ’ splice site is generally >45 nucleotides from the branch point The 3 ’ splice site is generally 18-38 nucleotides away from the metazoan branch point and 6-150 nucleotides from the yeast branch point

14 Pre-mRNA Splicing Requires ~100 proteins and 5 RNAs Occurs in a large RNP assembly known as the “ Spliceosome ” Catalytic component unknown but may be RNA-catalyzed Splicing via sequential transesterification reactions (same chemical steps as Group II intron splicing)


16 Pre-tRNA Splicing Splice

17 Splicing of Nuclear Pre-tRNA Introns (in Yeast ) Protein-catalyzed 1) endonuclease 2) ‘ligase’with 5 activities Endonuclease Ligase 3 ’ phosphodiesterase ‘ Kinase ’ ‘ Adenylase ’ ‘ 2'-Phospho transferase ’ ‘ Cyclic Phosphodiesterase ’ ‘ Ligase ’ OH P or ATP

18 Splicing in Archaea tRNAs and rRNAs Endonuclease: -symmetric homodimer - recognizes/cuts a bulge-helix-bulge motif formed by pairing of region near two exon- intron junctions Ligase: - joins exons and circularizes introns

19 Bulge-Helix-Bulge Motif Two 3 nt bulges on opposite strands separated by 4 bp Buldge Helix Buldge

20 tRNA Processing in Archaea BHB Endonuclease Ligase

21 rRNA Processing in Archaea

22 “ Inteins ” : Protein Splicing Too!

23 Catalytic Mechanisms: nucleophiles, introns, catalysts Splice-site Selection: splice junctions, recognition Summary of Intron Splicing Mechanisms

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