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Transcription 11/3/05

Transcription 11/3/05. Stable RNA. rRNA -Structural component of ribosomes tRNA-Adaptors, carry aa to ribosome Synthesis Promoter and terminator Post-transcriptional modification (RNA processing) Evidence Both have 5’ monophospates Both much smaller than primary transcript

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Transcription 11/3/05

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  1. Transcription 11/3/05

  2. Stable RNA • rRNA -Structural component of ribosomes • tRNA-Adaptors, carry aa to ribosome • Synthesis • Promoter and terminator • Post-transcriptional modification (RNA processing) • Evidence • Both have 5’ monophospates • Both much smaller than primary transcript • tRNA has unusual bases. EX pseudouridine

  3. tRNA and rRNA Processing • Both are excised from large primary transcripts • 1º transcript may contain several tRNA molecules, tRNA and rRNA • rRNAs simply excised from larger transcript • tRNAs modified extensively 5. Base modifications

  4. Examples of Covalent Modification of Nucleotides in tRNA 7-Methylguanylate (m7G) Inosinate (I) N6-Methyladenylate (m6A) N6-Isopentenyladenylate (i6A) Uridylate 5-oxyacetic acid (cmo5U) Dihydrouridylate (D) Pseudouridylate (Ψ) (ribose at C-5) 3-Methylcytidylate (m3C) Modifications are shown in blue. 5-Methylcytidylate (m5C) 2’-O-Methylated nucleotide (Nm)

  5. Eukaryotic Transcription • Regulation very complex • Three different pols • Distinguished by -amanitin sensitivity • Pol I—rRNA, least sensitive • Pol II– mRNA, most sensitive • Pol III– tRNA and 5R RNA moderately sensitive • Each polymerase recognizes a distinct promoter

  6. Eukaryotic RNA Polymerases

  7. Eukaryotic Polymerase I Promoters • RNA Polymerase I • Transcribes rRNA • Sequence not well conserved • Two elements • Core element- surrounds the transcription start site (-45 to + 20) • Upstream control element- between -156 and -107 upstream • Spacing affects strength of transcription

  8. Eukaryotic Polymerase II Promoters • Much more complicated • Two parts • Core promoter • Upstream element • Core promoter • TATA box at ~-30 bases • Initiator—on the transcription start site • Downstream element-further downstream • Many natural promoters lack recognizable versions of one or more of these sequences

  9. TATA-less Promoters • Some genes transcribed by RNA pol II lack the TATA box • Two types: • Housekeeping genes ( expressed constitutively). EX Nucleotide synthesis genes • Developmentally regulated genes. EX Homeotic genes that control fruit fly development. • Specialized (luxury) genes that encode cell-type specific proteins usually have a TATA-box

  10. mRNA Processing in Eukaryotes • Primary transcript much larger than finished product • Precursor and partially processed RNA called heterogeneous nuclear RNA (hnRNA) • Processing occurs in nucleus

  11. Capping mRNA • 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. • Guanosine cap is methylated. • First and second nucleosides in mRNA may be methylated BACK

  12. Polyadenylation • Polyadenylation occurs on the 3’ end of virtually all eukaryotic mRNAs. • Occurs after capping • Catalyzed by polyadenylate polymerase • Polyadenylation associated with mRNA half-life • Histones not polyadenylated

  13. Introns and Exons • Introns--Untranslated intervening sequences in mRNA • Exons– Translated sequences • Process-RNA splicing • Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete

  14. Splicing Overview • Occurs in the nucleus • hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP) • Primary transcripts assembled into hnRNP • Splicing occurs on spliceosomes consist of • Small nuclear ribonucleoproteins (SnRNPs) • components of spliceosomes • Contain small nuclear RNA (snRNA) • Many types of snRNA with different functions in the splicing process

  15. Splice Site Recognition • Introns contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule) • Internal intron sequences are highly variable even between closely related homologous genes. • Alternative splicing allows different proteins from a single original transcript

  16. Simplified Splicing Mechanism

  17. RNA pol III • Precursors to tRNAs,5SrRNA, other small RNAs • Promoter Type I • Lies completely within the transcribed region • 5SrRNA promoter split into 3 parts • tRNA promoters split into two parts • Polymerase II-like promoters • EX. snRNA • Lack internal promoter • Resembles pol II promoter in both sequence and position

  18. DNAse Footprinting • Use: promoter ID • End Label template strand • Add DNA binding protein • Digest with DNAse I • Remove protein • Separate on gel Protected region

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