Transcriptional Control

1
Transcriptional Control ?? Eukaryotes
2
Gene Expression ?? Eukaryotic Cells

• Control ???
• Spatial Temporal regulation
• Transcriptional control ??? DNA
• Alternate splicing ??? RNA
• Cytoplasmic control ??? mRNA stability
• Induction ??? transcriptional activity ???
  environment ??? biological factors

3
Spatial Temporal Regulation

• Spatial regulation ????????????? genes
  ???????????????????????????????? ??????????
  (tissue-specific expression) gene product
  ?????????????????????????????????????????
• Temporal regulation ????????????? genes
  ???????????????? ??????????????????
  ????????????????

4

• Transcriptional control ??? DNA RNA polymerase
  promoter
• Alternate splicing ??? RNA ????????? RNA
  ??????? (maturation ??? RNA)
• Induction ??? transcriptional activity
  ????????, ???, hormones ???????? genes ???????
  ??????????????????????? (development)

5
Cytoplasmic control ??? mRNA stability

• mRNA ??????????? ????????? transcription
  ?????????????????
• polypeptides ???????????????? gene
  ???????????????????
• polypeptides ??????????????????????????????????
• ??????? (degradation) mRNA ???????????????????????
  genes
• mRNA stable ??? Poly-A tail ??? Estrogen
• mRNA ???????????? 3 untranslated sequence AUUUA
  ????????????

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Transcriptional in Eukaryotes

• Factors ????????
• 1. RNA Polymerases 2. Promoters 3.
  Transcription Factors 4. Enhancers
• 5. Upstream promoter elements

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1. RNA Polymerase

• RNA polymerase ?? Eukaryotic cell ?? 3 types ???
  
• RNA Pol I ????????????? rRNA precursor (18S
  28S 5.8S)
• RNA Pol II ????????????? mRNA precursor
• RNA Pol III ????????????? tRAN precursor ???
• 5S rRNA precursor
• RNA polymerase ???? 3 ??????? eukaryote ?????
• s factor
• structure ???????
• ?? promoter ???????

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2. Promoter

• Promoter ?????? RNA Polymerase ????????? ???????
  ???
• Element(s) ???????? ???????????? DNA
  ?????????????????? RNA polymerase ?????
  (recognition site) ????????????????? promoter
  ???????? control ???????
• ??????? RNA polymerase ??????????? promoter ?????

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3. Transcription Factors

• Transcription factor ???? TF ??????????????? RNA
  Polymerase ???????????? ????
• TATA-binding protein (TBP)
• TFIIA --gt TFIIH
• ???????? DNA ???
• DNA binding domain ???
• Transcription activation domain

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4. Enhancers and Silencers

• ??????????? DNA ??????? stimulate transcription
  ??? promoter ????????????????? promoter
• ??????????????????????????????? DNA (fixed
  spatial relationship)????? control genes
  ????????????????????????? DNA

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Enhancer Silencer
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5. Upstream promoter elements

• ???? binding site ?????? transcription factors
  ??? RNA polymerase II
• ?????????????? promoter

13
Control ??? RNA Polymerase I?????? rRNA (18 S,
28 S ??? 5.8 S)

• Promoter ???????????? (control element) ???????
  2 blocks ???
• 1) Core element (Core)
• position ??????? - 45 ??? 20 ?????? RNA
  Polymerase
• 2) Upstream control element (UCE)
• position ??????? -156 ??? -107
• ???????? transcription ?????????????
• ???? 2 blocks ?????????? consensus sequence

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RNA Pol I Promoter

• Human rRNA promoter
• RNA Polymerase I ?????? -45 (downstream) ??? 20
  (upstream)
• TIF-I ?????? -67 (downstream) ??? -14 (upstream)
• ??????? RNA Pol I - TIF-I complex ????? promoter
• sequence ??? -67 ??? 20

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Control ??? RNA Polymerase II

• Promoter ?? control element 2 ??? ??????? genes
• 1. TATA box ???? Hogness box ??? -25
• ?? consensus sequence ??? TATAAAA
• ??????? upstream promoter element 21 bp
• (???? 3 repeats) ??????? -40 ??? -103
• 2. CAAT box
• consensus sequence (GC)CCAATCT ??? -75

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Control Elements ??? RNA Polymerase II Promoter
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• Initiation complex ??? RNA polymerase II
• ??????? TBP ??? TFII A --gt H

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• Enhancer ??? RNA pol II
• Stimulate transcription ??? promoter?????
  consensus sequences
• ???? tissue-specific ???? mouse immunoglobulin
  enhancer ????????? immunoglobulin genes ???????
  immune system ??????????????? mouse connective
  tissue cell

Enhancer
Closed promoter
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Enhancer

• Enhancer ???? RNA polymerase ???? promoter
  ??????? transcription

Opened Promoter
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Control ??? RNA Polymerase III

• Promoter ???? Internal promoter
• 5S rRNA gene promoter
• ?? control element upstream ?????????? 55 ???
  80
• tRNA gene promoter
• ?? 2 blocks ?????? A block ??????? 8 ??? 30
  ???
• B block ??????? 51 ??? 72

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Posttranscriptional Control ???? RNA Processing
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Posttranscriptional Control in Eukaryote

• RNA ??? ?????????????? nucleus ???????? Primary
  transcript
• ??????????????????????????????????????
• Primary transcript ???? precursor ??? mRNA
  ???????? Heterogeneous nuclear RNA (hnRNA)

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Heterogeneous nuclear RNA (hnRNA)
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mRNA Processing

• hnRNA ????????? modified ????????? 3 ???????
  ???
• 1. Capping
• 2. Tailing
• 3. Intron Splicing

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1. Capping

• ???????????????? 5 ??????? Cap ????????
  7-methylguanosine
• ????????????????? nuclease
• ??? ribosome ?? mRNA ??? ???
• ???????? mRNA ?????? nucleus ?? cytoplasm
• Guanine triphosphonucleotide
• (GTP) ?????? nucleotide ???? 5
• Guanine (G) ??????? CH3(methyl) ??????? N7
  ??????????

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Capping Methylation
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Capping Reaction
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2. Tailing

• ?????????????? Adenine ??????????????????? 3
  end (trailer) ??? hnRNA ???????? Polyadenylation
  
• ??? poly-A polymerase
• ????? stability ??? transportation RNA ??????
  nucleus

29
Tailing ??? mRNA
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3. Intron splicing

• Primary transcript ??? mRNA, tRNA ??? rRNA
  ???????????????????????????? ??????? mature RNA
  ?????????? cytoplasm ???
• ???????? ???? noncoding sequence ???????? Intron
  ??????????? coding sequences ???????????? Exon

31
(No Transcript)
32

• mRNA
• Splicing

33
Intron ???????? 4 ?????

• 1. Group I intron intron ?? 5 UA ??? 3
  G
• nuclear rRNA precursor ??? mitochondrail rRNA
• 2. Group II intron intron ?? 5 G ??? 3
  A-Nn-G
• mitochondrial mRNA precursor
• 3. Nuclear mRNA intron intron ?? 5 G ??? 3
  UACUAAC box (TACTAAC box ?? DNA)
• mRNA precursor ?? nucleus ??? eukaryotes
• 4. tRNA intron ??????????????

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Mechanism ??? Splicing

• 1. Self splicing ??? Autocatalytic property of
  intron ???
• 1.1 ??????? Guanine-containing nucleotide
• (GMP, GDP, GTP) ???? group I introns
• 1.1 ?????????? Guanine-containing nucleotide
• ???? group II introns

35

• 2. Spliceosome ??????? snRNA ??????????????
  ????? proteins ?????????????? ??????????? small
  nuclear ribonucleoproteins (snRNPs) ???? Snurps
  ???? Spliceosomes ???? mRNA precursor
• 3. tRNA precursor splicing ??? Splicing
  endonuclease ??? Splicing ligase

36
Self-splicing ??? Group I intron??????? Guanine
37
Self-splicing ??? Group II intron??????????
Guanine
38
Spliceosome??????? Snurps ??? snRNA ?? enzyme
activity (Ribozyme)
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Alternative Splicing ??? mRNAs

• ?????? Introns ???? ??? Exons ???????????? mRNA
  precursor
• ????????? Splicing site ??? 5 ??? 3 ?? intron
  ????????????? ????? gene 1 gene ????????? mRNA
  ??????????? Family ??? mRNAs
• ??????? 1 gene ?????? code ??? protein
  ??????????????????????? ???????????? Protein
  Isoforms
• ???? Immunoglibulin genes code ??? antibody
  ????????

40
Alternate Splicing
41
tRNA Splicing

• Splicing endonuclease ??????????? 2 ???????
  intron
• Splicing ligase ?????????? 2 exons ???????????

42
Reverse Transcription
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Reverse transcription

• ?????????? DNA ?????? RNA ?????????? ???
• ??? RNA-dependent DNA polymerase ????
• Reverse transcriptase (RT)
• DNA ?????????????? Complementary DNA (cDNA)
• RNA tumor virus ????????????????????????? cDNA
  ????
• ?????????? (integrate) ???? DNA ??? host ???
• cDNA ??????????????????? mRNA ?????????????
• (in vitro)

44
Reverse Transcription
45
Mechanism ??? Reverse Transcription
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Central Dogma Update
V. 2
V. 1
V. 3
V. 4
(forbidden)