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 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

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

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

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

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

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

Spliceosomal intron removal Consensus sequences at splice sites in vertebrates GU at 5' endAG 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”)

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