【正文】 s: Howard Temin knew that retrovirus genomes were posed of RNA and observed that replication was inhibited by actinomycin D therefore he proposed the concept of reverse transcription ( Nobel prize awarded to Baltimore and Temin, 1975 ). 1970: Temin and David Baltimore (separately) discover the RNAdirected DNA polymerase reverse trascriptase ? Retroviruses Retrotranscriptase Three enzymatic activities: RDNAP, DNAP and RNase H ? cDNA See you next time! 演講完畢，謝謝觀看！ 。 RNA ligase。 junctions on different RNA molecules ? Relatively rare otherwise ? Occurs naturally in trypanosomes and in C. elegans maybe elsewhere too. Alternative splicing can generate multiple proteins from a single gene Capping Tailing Posttranscriptional processing of prerRNA ? In karyotes Cleavage, trimming and modification ? In eukaryotes 1) Cleavage, trimming and modification 2) Splicing (Some eukaryotes such as Tetrahymena四膜蟲 ) Selfsplicing of PrerRNA ? Discovery (1989 Nobel Prize Laureate in Chemistry) found 26S rRNA has one 400 b intron in some Tetrahymena strains in 1981:This Intron can SelfSplice。Polyadenylation A poly(A) tail is added to the 3’ end of the transcript in 2 steps… 1) cleavage: the RNA is cut 1030 nucleotides downstream of a specific sequence in the 3’UTR 2) addition of A’s (100200 are added) to generate a poly(A) tail Coding sequence of the gene TATA box +1 2530 bp mRNA Translated region 5’UTR 3’UTR ATG Stop mRNA 5’ CAP AAUAAA CPSF 1) CPSF = “cleavage/polyadenylation specificity factor” (3 subunits) recognizes the AAUAAA sequence in the RNA 3’UTR and binds to it 2) Recruits CFI and CFII = “cleavage factors” 3) Recruits PAP = “poly(A) polymerase” CFI CFII PAP A ribonucleoprotein plex mRNA 5’ CAP AAUAAA CPSF ? The RNA is cleaved 1030 nucleotides downstream of the AAUAAA by CFI/II ? This generates a 3’OH to which PAP adds A residues. It doesn’t copy from a template just needs a 3’OH of an RNA ? The poly(A) tail is bound by poly(A)binding protein CFI CFII PAP AAUAAA AAAAAAAAAAAAA100200 Why have a Poly(A) tail? ? The poly(A) tail promotes stability of the mRNA (prevents degradation by 3’ exonucleases) ? The poly(A) tail promotes translation of the mRNA ? A ribosomal protein subunit that interacts with the CAP structure is stimulated in CAP binding by poly(A) binding protein Therefore the CAP and the poly(A) tail work synergistically to recruit mRNA to the ribosome ? Create stop codon ? Alternative tailing provides the possibility one gene, two or more proteins Eukaryotic Genes are Split ? Discover: Richard J. Roberts and Phillip A. Sharp( The Nobel Prize laureate in Physiology or Medicine 1993) ? Introns intervene between exons ? Examples: actin gene has 309bp intron separates first three amino acids and the other 350 or so ? But chicken proalpha2 collagen gene is 40kbp long, with 51 exons of only 5 kbp total. ? The exons range in size from 45 to 249 bases ? Mechanism by which introns are excised and exons are spliced together is plex and must be precise Denature Hybridize with mature mRNA And visualize under EM Rloop techniques DNA template strand Mature mRNA Splicing of PremRNA ? Splicing signals “internal cause” ? snRNPs “external cause” ? Splicing reaction two Transesterification reactions ? Assembly of spliceosomes Sequence requirements for splicing G 3’ Exon 5’ Exon AG GU AG Intron 18 40 nucleotides 3. Branch point YNYRAY Y= pyrimidine R= purine N = anything 1. 5’ splice site 2. 3’ splice site Splicing occurs through two Transesterification reactions P O O O O X Y RNA Chain + R OH P O O O O R Y X OH + ? In a transesterification reaction a phosphodiester bond is transferred to a di