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Transcription. Lecture 6 of Introduction to Molecular Biology 生理所 蔡少正. Enzymatic Synthesis of RNA. Precursors in RNA synthesis: rNTPs Free 3’OH react with 5’ phosphate of rNTPs Complimentary to DNA minus strand RNA chain grows from 5’ to 3’ direction Catalyzed by RNA polymerase

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  1. Transcription Lecture 6 of Introduction to Molecular Biology 生理所 蔡少正

  2. Enzymatic Synthesis of RNA • Precursors in RNA synthesis: rNTPs • Free 3’OH react with 5’ phosphate of rNTPs • Complimentary to DNA minus strand • RNA chain grows from 5’ to 3’ direction • Catalyzed by RNA polymerase • Four steps: binding of RNA polymersae to promoter, initiation, chain elongation, and termination

  3. RNA Polymerases --- Common: complex structure containing multiple subunits (two large subunits [>100kD] and several small subunits); do not need primer for initiation --- Differences:

  4. Promoter(I) A region of DNA involved in binding of RNA polymerase to initiate transcription. TATA box: (TATAA/TAA/T) 25 bp upstream of transcription start site. locating transcription initiation site, maintain promoter activity CATA box TATA less promoter: GC box (house-keeping genes; developmentally regulated genes)

  5. Promoter(II) Upstream element: (1) GC box (CCGCCC): usually multiple copies; orientation independent. (2) Cat box (CCAAT): CCAAT-binding transcription factor (CTF) CCAAT/enhancer binding protein (C/EBP) Initiator: (PyPyANT/APyPy) conserved sequences around the transcription start site; required for optional transcription activity Downstream element:

  6. General Transcription Factors Definition: combine with RNA polymerase to form a preinitiation complex that is competent to initiate transcription as soon as nucleotides are available Class II Factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH TBP (TATA box binding protein)

  7. Enhancer A cis-acting sequence that increases the utilization of eukaryotic promoters; can function in either orientation and in any location (upstream or downstream) relative to the promoter.

  8. Termination: (a) RNA polymerase I: Termination occurs at a discrete site > 1000 bp downstream of the mature 3' end, which is generated by cleavage. Termination involves recognition of an 18 bp terminator sequence by an ancillary factor. (b) RNA polymerase III: Termination usually occurs at a U run (U4 or U5) within a G-C rich region. (c) RNA polymerase II: Termination involves a consensus sequence AAUAAA for cleavage to generate a 3' OH end for polyadenylation and some small RNAs (e.g. U7 sn RNA).

  9. Post-transcriptional Modification (RNA processing): All the RNA molecules are modified after transcription. Both tRNA and rRNA molecules are excised from large primary transcripts. Transcription

  10. Capping The 5' terminus of a mRNA molecule carries a methylated guanosine derivative, 7-methylguanosine (7-MeG), in an unusual 5'-5' linkage to the 5'-terminal nucleotide of the primary transcript, astructure called “Cap”. Function of Capping Increase mRNA stability Enhance mRNA translation Transport of RNA out of nucleus Proper splicing of pre-mRNA

  11. Polyadenylation Most animal mRNA are terminated at the 3’ end with a poly(A) tract, which is added by poly(A) polymerase. The length of poly(A) segment can be from 50 to 250 nucleotides long. Polyadenylation signal: AAUAAA With poly(A) No poly(A)

  12. RNA Splicing 1. Most higher eukaryotic genes coding for mRNA and tRNA (few rRNA) are interrupted by unrelated regions called introns. 2. mRNA has 0-60 introns while tRNA has 0 or 1 intron Transcription start site Gene E1 I1 E2 I2 E3 hn RNA: Primary transcript mRNA: Mature transcript

  13. Splicing Signals The GU-AG rule: Get-U(you)-And-Go Consensus Sequences in Mammals: 5’-AG/GUAAGU--intron--YNCURAC--YNAG/G3’

  14. A Signal at the Branch Yeast: UACUAAC Higher Eukaryotes: U47NC63U53R72A91C47

  15. SnRNPs Small nuclear RNA (snRNA): the agents that recognize the splicing signals • The SnRNAs are coupled with proteins in complex called snRNPs • U1 snRNA: Bind 5’ splice site of an mRNA precursor: necessary but not sufficient for splicing • U6 snRNP: Associated with the 5’ end of intron by base pairing; essential for splicing process • U5 snRNP brings together the last nucleotide in one exon and the first nucleotide of next exon • U4 snRNP base-pair with U6 until U6 is needed for splicing

  16. Formation of Spliceosome

  17. Alternative Splicing Occurs every 1 in 20 genes Significance of alternative splicing: --- Localization of protein: member-bound vs. secreted form proteins --- Tissue specific expression --- Different signaling pathway --- Sex differentiation --- Increase protein diversity

  18. Self Splicing of Group I & II Introns Group I intron can be removed without help from other protein Group II intron self-splice using an A-branch lariat intermediate

  19. RNA Editing Trypanosomatid mitochondria encode incomplete mRNAs that must be edited before they can be translated Editing occurs in the 3’ to 5’ direction by successive action of one or more guide RNAs

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