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Posttranscriptional Modification of DNA

Posttranscriptional Modification of DNA. Primary Transcript – newly synthesized RNA Mature tRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts In prokaryotes, 1 o transcripts often contain several tRNA precursors

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Posttranscriptional Modification of DNA

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  1. Posttranscriptional Modification of DNA • Primary Transcript – newly synthesized RNA • Mature tRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts • In prokaryotes, 1o transcripts often contain several tRNA precursors • Ribonucleases (RNases) cleave the large primary transcripts to their mature lengths • RNase P – cleaves 5' end • RNase D – cleaves 3' end • tRNA nucleotidyl transferase adds CCA to 3' end • May have segments removed • May have bases further modified

  2. Ribosomal RNA Processing • Ribosomal RNA in all organisms are produced as large primary transcripts that require processing • Processing includes methylation and cleavage by endonucleases • Prokaryotic rRNA primary transcripts ~30S • Contain one copy each: 16S, 23S, 5S rRNA Endonucleolytic cleavage of rRNA precursors in E. coli

  3. Eukaryotic mRNA Processing • In prokaryotes the most primary mRNA transcript is translated directly • In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm • Eukaryotic mRNA is processed in the nucleus without interfering with translation • In some mRNA, pieces are removed from the middle and the ends joined (splicing) Modification of Ends • All eukaryotic mRNA precursors undergo modifications to increase their stability and make them better substrates for translation • Ends are modified so they are no longer susceptible to exonuclease degradation • The 5' ends are modified before the mRNA precursors are completely synthesized

  4. Formation of a guanosine cap at the 5' end of a eukaryotic mRNA precursor. Guanylate base is methylated at N-7 2- Hydroxyl groups of last two riboses may also be methylated

  5. Poly A tails at the 3' ends of mRNA precursors • Eukaryotic mRNA precursors are also modified at their 3' ends • On gene (DNA) this is the poly(A) site • A poly A polymerase adds up to 250 adenylate residues to the 3' end of the mRNA precursor • This poly A tail is progressively shortened by 3' exonucleases • The poly A tail increases the time required for nucleases to reach the coding region Some mRNA Precursors are Spliced • Introns - internal sequences that are removed from the primary RNA transcript • Exons - sequences that are present in the primary transcript and the mature mRNA • Splice sites - junctions of the introns and exons where mRNA precursor is cut and joined

  6. Triose phosphate isomerase gene (9 exons and 8 introns) • 4 classes of introns - Third group found only in eukaryotes • Groups I and II are self-splicing – no ATP required for energy • Third group (largest class) – spliceosomal introns • Splicing catalyzed by spliceosome protein complex • Lariat formation mechanism (see following slide) • Fourth class – requires ATP and an endonuclease

  7. Spliceosomal intron removalConsensus sequences at splice sites in vertebrates GU at 5' end AG at 3' end • The spliceosome is a large RNA-protein complex which catalyzes splicing reactions • It contains 45 proteins and 5 small nuclear RNA (snRNA) molecules (“snurps”)

  8. Splicing allows for variation in RNA product from the same gene • Length can vary depending on poly(A) site • Splicing pattern for intron removal generates different mRNAs

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