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RNA ’ s

RNA ’ s. protein. DNA. RNA. cDNA. EST (Expressed Seq. Tag). Non-coding RNAs. Protein coding RNA – mRNA Non-coding RNAs tRNA (transfer RNA) rRNA (ribosomal RNA) Involved in protein synthesis 80-85% of total RNAs snRNA (small nuclear RNA) Localized to the nucleus

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RNA ’ s

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  1. RNA’s

  2. protein DNA RNA cDNA EST (Expressed Seq. Tag)

  3. Non-coding RNAs • Protein coding RNA – mRNA • Non-coding RNAs • tRNA (transfer RNA) • rRNA (ribosomal RNA) • Involved in protein synthesis • 80-85% of total RNAs • snRNA (small nuclear RNA) • Localized to the nucleus • Consists of families of RNAs responsible for functions such as RNA splicing • E.g. splicsome – five snRNAs U1, U2, U4, U5 and U6 • snoRNA (small nucleolar RNA) • Synthesis of rRNA occurs in nucleolus, specialized structure within the nucleus, facilitated by snoRNAs • miRNA (micro RNA) • Short 22-nt, regulating gene expressions

  4. mRNA (messenger RNA)

  5. mRNA (messenger RNA) – another view

  6. Transcription • Gene sequence is copied from one strand • Sense strand = mRNA sequence • Antisense strand is used to generate mRNA sequence • 5’CGCTATAGCGTTTCAT 3’ -- antisense, template strand • 3’GCGATATCGCAAAGTA 5’ – sense, coding strand

  7. Template, anti-sense sense

  8. Transcription initiation • Double-helix DNA strands are separated in the gene coding region • Which enzyme detects the beginning of a gene ? • RNA Polymerase (multi-subunit enzyme that synthesize RNA) binds to promoter • RNA polymerase I – 28S, 5.8S and 18S rRNA genes • RNA polymerase II – coding genes, snRNA • RNA polymerase III – tRNA, 5S rRNA, snoRNA • Other enzymes • General (Basal) Transcription Factor (GTF) • TFIIA, TFIIB, TFIID • TFIID – recognize promoter sequence

  9. Promoter in E.coli

  10. Transcription initiation in E.coli

  11. Transcription initiation in eukaryotes • Promoter consists of • -25 or TATA box(TATAWAW; W=A, T) • And Inr (initiator) seq. (YYCARR: Y=C,T; R=A,G)

  12. Transcription initiation in eukaryotes Initial contact is made by general transcription factor (GTF) TFIID, which consists of TATA-binding protein (TBP) and at least 12 TBP-associated factors (TAF)

  13. Transcription Start Site (TSS) • www.cs.uml.edu/~Kim/580/review_polII_11_Kadonaga.pdf • TSS – the first base copied to mRNA • Core promoter – region around a TSS • Conventionally, core promoter has • TA box at -30 bp of a Inr (Initiator) • Transcription Factor (TF) bind to TATA box, Inr sequence, and other sites; bend DNA 90 degree; recruite general TF • CpG islands: 300-3000 bp of C & G in 40% of promoters • More recently, • TATA box only in 10-20% or promoters

  14. Core Promoter Elements • IIB Recognition Element (BRE) • BREu (BREd) suppresses (enhances) transcription • TATA box – TATAWAAR (metazoans) • W (A,T); R (A,G-Purine); Y (T,C – Pyrimidine) • Inr – YYANWYY (A+1) • DPE (downstream Core Promoter Element) • MTE (Motif Ten Element)

  15. Focused/Dispersed TSS • Focused (Sharp) TSS • Distinct TSS site • Usually TATA box in sharp TSS • Primarily in tissue-specific expressions • Dispersed (Broad) TSS • Multiple weak start sites in 50-100 nt • A few Inr or Inr-like seq in the neighborhood • Generally associated with ubiquitously expressed genes • Thought to be related to CpG islands

  16. How to recognize the end of transcription ? • Terminator seq. stalls polymerase

  17. Splicing • Alternative splicing to produce mRNA • Splicesome – a collection of snRNA

  18. Function of Introns • www.cs.uml.edu/~kim/580/review_intron.pdf • When inserted into protomer, boost expression level • First introns are long • Alternative exons are flanked by long introns • But, association between intron length and expression breadth in human is not found • Removal of 2nd intron of human beta-globin gene reduces the efficiency of 3’-end formation • RNA pol II elongation rate – 3.8kb/min • Introns may serve as time delays between activation of a gene

  19. Protein Synthesis • Translation from mRNA to protein • mRNA is transported out of a cell nucleus, translated • tRNA (transfer RNA)

  20. AA is attached in upper end (blue) tRNA is bound to mRNA at bottom (green)

  21. Ribosomes • Role of ribosomes in protein synthesis • Coordinate protein synthesis by placing mRNA, tRNA and protein factors in their correct positions • Components of ribomsomes catalyze at least some chemical reactions occurring during translation • Each ribosome consists of two units • 45S (18S, 28S, 5.8S) subunits in eukaryotes • 50S and 30S in bacteria (16S, 23S, 5S)

  22. Schematic drawing of secondary structure for 16S rRNA. Intrachain folding pattern includes loops and double-stranded regions.

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