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13 주차 수업자료 PowerPoint Presentation
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13 주차 수업자료

13 주차 수업자료

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13 주차 수업자료

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  1. 13주차 수업자료

  2. Chapter 18. Regulation at the RNA level

  3. RNA regulation • Degradation rate of mRNA • Modification of untranslatable RNA to be translated • mRNA translation • Binding of antisense RNA to mRNA • Riboswitch (part of mRNA that can bind small target molecule) • Preferential translation

  4. Degradation of mRNA • CsrABC (Csr: carbohydrate storage regulator) regulatory system in bacteria • CsrA: RNA-binding protein • CsrB, CsrC: non-coding regulatory RNA (ncRNA) or small RNA (sRNA) • Controls balance between glycogen synthesis (repressed by CsrA) and glycolysis (activated by CsrA) • CsrA also binds to glg mRNA (genes involved in glycogen synthesis) and promotes degradation • Increased ribonuclease attack? • Inhibit translation  not protected by ribosome  faster degradation?

  5. Cleavage of mRNA for translation • Prokaryote: primary transcript = mRNA • Eukaryote: primary transcript + cap + polyA tail = mRNA • adhE mRNA must be processed by RNaseIII to reveal RBS (ribosome binding site) for translation into alcohol dehydrogenase • RNaseIII also cleaves tRNA and rRNA precursors • adhE mRNA is also controlled by degradation by RNaseG

  6. Repression of translation by regulatory protein • Ferritin: Fe storage protein • IRE (iron-responsive element): 5’-UTR of ferritin mRNA, stem-and-loop • Low Fe: IRP (iron regulatory protein) binds to IRE  translation X • High Fe: no IRP binding  translation O • Aconitase/IRP1: Fe4S4 cluster is required for enzyme activity, converts citrate to isocitrate (in Krebs cycle) • Low Fe: one Fe lost from Fe4S4cluster  IRP1 changes conformation  RNA-binding translation repressor • High Fe: IRP1 active as aconitase

  7. Regulation of ribosomal protein synthesis in bacteria by translational repression • Auto-regulation • Ribosomal protein preferentially binds to rRNA • If no more rRNA is available, ribosomal protein binds to its own mRNA and blocks RBS (ribosome binding site)  translation X

  8. Activation of translation by regulatory protein • Light  protein synthesis in chloroplast: How to control? • Rubisco (ribulose bisphosphate carboxylase): CO2 fixation during photosynthesis • Translational activator: binds to A-rich region in 5’-UTR • cPABP (chloroplast polyadenylate binding protein): light  electron transfer  reduced sulfhydryl group of cPABP  bind mRNA  activate translation

  9. Regulation of translation by antisense RNA • mRNA = sense RNA  complementary  antisense RNA • How is the antisense RNA generated??? --- “anti-gene” • Bacterioferritin: Fe storage protein in bacteria (required when Fe level is high) • Fur (ferric uptake regulator): iron level sensor • If Fe level is low, activate anti-bfr gene  suppress the production of bacterioferritin • If Fe level is high, repress anti-bfr gene  production of bacterioferritin

  10. Regulation of translation by ribosome modification • Proliferation signal  phosphorylation of S6 protein (in ribosomal small subunit) • Preferentially translate mRNA containing 5’-TOP (5’-terminal oligopyrimidine tract) • What mRNA? • Ribosomal protein • Elongation factor

  11. RNA interference (RNAi) • Gene silencing induced by dsRNA • Defense mechanism against virus (infection by RNA virus  dsRNA intermediate) • Dicer cleaves dsRNA into 21-23 bp siRNA (small interfering RNA)  bound by RISC (RNA-induced silencing complex)  strand separation and degradation of single strand by RISC (slicer activity by Argonaut protein, AGO) recognize target RNA and cleavage by RISC

  12. Regulation by micro RNA (miRNA) • miRNA • Precursor mRNA is about 70 nt long • Precursor forms stem-and-loop • 1-3 unpaired bp in the middle • Strand separation by RISC • Block translation of mRNA • siRNA • No unpaired bp, complete bp • Promote degradation of mRNA with cleavage by RISC • piRNA • Piwi-interacting RNA • Suppression of transposon activity in germ cells

  13. Structure of dicer • dsRNA binding domain, 2 RNase III domains, and PAZ domain • PAZ domain prefers 2 nt overhang on the 3’ end of RNA • Dicer: molecular ruler

  14. Argonaute proteins and putative models for target-RNA recognition • AGO, PIWI, WAGO subfamily • PAZ domain binds 3’ end of guide strand • Phosphate binding pocket located between MID and PIWI domains binds 5’ end of guide strand • miRNA: fixed-end model • siRNA: two-state model Nature, 457, Jan 2009

  15. Biogenesis and mechanism of action of the main classes of small regulatory RNA Nature, 457, Jan 2009

  16. Key proteins in RNA silencing in various organisms Nature, 457, Jan 2009

  17. Amplification of RNAi by RdRP • 50 siRNA can silence thousands of target RNA  How is this possible??? • RNA-dependent RNA polymerase (RdRP) • No primer is required • RNAi can also spread from cell to cell (in plants and in C. elegance) • But, mammals do not possess RdRP responsible for RNAi amplification, possibly due to the development of specific immune system

  18. Experimental administration of siRNA • Introduction of long dsRNA, ss antisense RNA, short 21-23 nt dsRNA • Single DNA segment (single promoter) generating stem-and-loop • Single DNA segment flanked by two opposing promoters • Two DNA segments (inverse each other) having separate promoters

  19. CRISPR: anti-viral defense in bacteria

  20. Transcriptional attenuation by premature termination • Leader region (1,2,3,4) • in front of structural gene • forms stem-and-loop (1-2 & 3-4, or 2-3 when 1 is bound by protein) • Terminator loop vs. anti-terminator (pre-emptor) loop • In E. coli, leader region (for leader peptide) is bound by ribosome • Corresponding aa shortage: pre-emptor (2-3) forms  transcription by RNA pol continues • Corresponding aa abundance: terminator (3-4) forms  prevent further elongation by RNA pol

  21. Attenuation protein and riboswitch • In Bacillus, no ribosome binding and no leader peptide  Trp attenuation protein (TRAP, RNA-binding protein) binds trp mRNA leader region in the presence of Trp  stem-and-loop  promotes termination • Riboswitch: domain of mRNA that can interact with small molecules and control gene expression by altering stem-and-loop structure  attenuation mechanism and translational inhibition mechanism

  22. RNA thermosensor • Riboswitchthat responds to temperature • At low temp  SD sequence in stem-and-loop  no translation (ex. rpoH mRNA) • high temp  stem-and-loop destabilize  ribosome can bind to SD sequence  translation (ex. rpoH mRNA)

  23. Nomenclature Table