1 Gene Expression Eukaryotic Cells Control Spatial Temporal

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Gene Expression �� Eukaryotic Cells Ø Control ��� Spatial & Temporal regulation F Transcriptional

Gene Expression �� Eukaryotic Cells Ø Control ��� Spatial & Temporal regulation F Transcriptional control ��� DNA F Alternate splicing ��� RNA F Cytoplasmic control ��� m. RNA stability F Induction ��� transcriptional activity ��� environment ��� biological factors F 2

Spatial & Temporal Regulation Spatial regulation: ������ genes ��������� (tissue-specific expression) gene product ���������

Spatial & Temporal Regulation Spatial regulation: ������ genes ��������� (tissue-specific expression) gene product ��������� Ø Temporal regulation: ������ genes 3 ������� Ø

Transcriptional control ��� DNA ~ RNA polymerase & promoter Ø Alternate splicing ��� RNA

Transcriptional control ��� DNA ~ RNA polymerase & promoter Ø Alternate splicing ��� RNA ~ ���� RNA ������� (maturation ��� RNA( Ø Induction ��� transcriptional activity ~ ���� , hormones ������ genes ������������ (development( 4 Ø

Cytoplasmic control ��� m. RNA stability m. RNA ������ transcription ������� Ø polypeptides �������

Cytoplasmic control ��� m. RNA stability m. RNA ������ transcription ������� Ø polypeptides ������� gene ��������� Ø polypeptides ��������� Ø ������� (degradation) m. RNA ��������� genes Ø m. RNA stable ��� Poly-A tail ��� Estrogen Ø m. RNA ������ 3’ untranslated 5 Ø

Transcriptional in Eukaryotes Factors ����. 1 RNA Polymerases 2. Promoters 3. Transcription Factors 4.

Transcriptional in Eukaryotes Factors ����. 1 RNA Polymerases 2. Promoters 3. Transcription Factors 4. Enhancers. 5 Upstream promoter elements 6

. 1 RNA Polymerase RNA polymerase �� Eukaryotic cell �� 3 types ��� RNA

. 1 RNA Polymerase RNA polymerase �� Eukaryotic cell �� 3 types ��� RNA Pol I ������� r. RNA precursor (18 S + 28 S + 5. 8 S) RNA Pol II ������� m. RNA precursor RNA Pol III ������� t. RAN precursor �� 5 S r. RNA precursor Ø RNA polymerase ���� 3 ������� eukaryote ����� Ø - s factor – structure ������� 7

. 3 Transcription Factors Ø Transcription factor ���� TF �������� RNA Polymerase ������ TATA-binding

. 3 Transcription Factors Ø Transcription factor ���� TF �������� RNA Polymerase ������ TATA-binding protein (TBP) F TFIIA --> TFIIH F Ø ���� DNA binding domain ��� F Transcription activation domain F 9

. 4 Enhancers and Silencers Ø Ø ������ DNA ������� stimulate transcription ��� promoter

. 4 Enhancers and Silencers Ø Ø ������ DNA ������� stimulate transcription ��� promoter ���������������� DNA (fixed spatial relationship)����� control genes 10

Enhancer & Silencer 11

Enhancer & Silencer 11

. 5 Upstream promoter elements ���� binding site ������ transcription factors ��� RNA polymerase

. 5 Upstream promoter elements ���� binding site ������ transcription factors ��� RNA polymerase II Ø ������� promoter Ø 12

Control ��� RNA Polymerase I ������ r. RNA (18 S, 28 S ��� 5.

Control ��� RNA Polymerase I ������ r. RNA (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 13 �������

RNA Pol I Promoter Human r. RNA promoter: RNA Polymerase I ������ -45 (downstream)

RNA Pol I Promoter Human r. RNA promoter: RNA Polymerase I ������ -45 (downstream) ��� +20 (upstream) Ø TIF-I ������ -67 (downstream) ��� -14 (upstream) Ø ������� RNA Pol I - TIF-I complex ���� promoter 14 Ø

Control ��� RNA Polymerase II Promoter: �� control element 2 ������� genes 1. TATA

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 15

Control Elements ��� RNA Polymerase II Promoter 16

Control Elements ��� RNA Polymerase II Promoter 16

Initiation complex ��� RNA polymerase II Ø ������� TBP ��� TFII A --> H

Initiation complex ��� RNA polymerase II Ø ������� TBP ��� TFII A --> H Ø 17

Enhancer ��� RNA pol II: Ø Stimulate transcription ��� promoter����� consensus sequences Ø ����

Enhancer ��� RNA pol II: Ø Stimulate transcription ��� promoter����� consensus sequences Ø ���� tissue-specific ���� mouse immunoglobulin enhancer ����� immunoglobulin genes ������� immune system �������� mouse. Enhancer connective tissue cell Closed promoter 18

Enhancer Ø Enhancer ���� RNA polymerase ���� promoter ������� transcription Opened Promoter 19

Enhancer Ø Enhancer ���� RNA polymerase ���� promoter ������� transcription Opened Promoter 19

Control ��� RNA Polymerase III Promoter: ���� Internal promoter Ø 5 S r. RNA

Control ��� RNA Polymerase III Promoter: ���� Internal promoter Ø 5 S r. RNA gene promoter : �� control element upstream ������� +55 ��� +80 Ø Ø t. RNA gene promoter �� 2 blocks ������ A block ������� +8 ��� +30 ��� 20

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Posttranscriptional Control in Eukaryote Ø Ø Ø RNA �������� �� nucleus ���� Primary transcript

Posttranscriptional Control in Eukaryote Ø Ø Ø RNA �������� �� nucleus ���� Primary transcript �������� Primary transcript ���� precursor ��� m. RNA ���� Heterogeneous nuclear 22

Heterogeneous nuclear RNA (hn. RNA) 23

Heterogeneous nuclear RNA (hn. RNA) 23

m. RNA Processing Ø hn. RNA ����� modified ����� 3 ������� 1. Capping 2.

m. RNA Processing Ø hn. RNA ����� modified ����� 3 ������� 1. Capping 2. Tailing 3. Intron Splicing 24

Capping & Methylation 26

Capping & Methylation 26

Capping Reaction 27

Capping Reaction 27

. 2 Tailing Ø Ø Ø ������� Adenine �������� 3’ end (trailer) ��� hn.

. 2 Tailing Ø Ø Ø ������� Adenine �������� 3’ end (trailer) ��� hn. RNA ���� Polyadenylation ��� poly-A polymerase ����� stability ��� transportation RNA ������ nucleus 28

Tailing ��� m. RNA 29

Tailing ��� m. RNA 29

3. Intron splicing Ø Primary transcript ��� m. RNA, t. RNA ��� r. RNA

3. Intron splicing Ø Primary transcript ��� m. RNA, t. RNA ��� r. RNA ��������� �� ������� mature RNA ������� cytoplasm ��� Ø ���� noncoding sequence ���� Intron ������ coding sequences ������ Exon 30

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m. RNA Splicing 32

m. RNA Splicing 32

Intron ���� 4 �����. 1 Group I intron : intron �� 5’ UA ���

Intron ���� 4 �����. 1 Group I intron : intron �� 5’ UA ��� 3’ G F nuclear r. RNA precursor ��� mitochondrail r. RNA . 2 Group II intron : intron ��’ 5 G ��� 3’ ANn-G F mitochondrial m. RNA precursor . 3 Nuclear m. RNA intron : intron ��’ 5 G ��� 3’ UACUAAC box (TACTAAC box �� DNA( F m. RNA precursor �� nucleus ��� eukaryotes 4. t. RNA intron : �������� 33

Mechanism ��� Splicing 1. Self splicing ��� Autocatalytic property of intron ��� 1. 1

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 34

2. Spliceosome: ������� sn. RNA ������� proteins ������� small nuclear ribonucleoproteins (sn. RNPs) ����

2. Spliceosome: ������� sn. RNA ������� proteins ������� small nuclear ribonucleoproteins (sn. RNPs) ���� Snurps ���� Spliceosomes ���� m. RNA precursor 3. t. RNA precursor splicing: ��� Splicing endonuclease ��� Splicing ligase 35

Self-splicing ��� Group I intron ������� Guanine 36

Self-splicing ��� Group I intron ������� Guanine 36

Self-splicing ��� Group II intron ����� Guanine 37

Self-splicing ��� Group II intron ����� Guanine 37

Spliceosome ������� Snurps ��� sn. RNA �� enzyme activity (Ribozyme) 38

Spliceosome ������� Snurps ��� sn. RNA �� enzyme activity (Ribozyme) 38

Alternate Splicing 40

Alternate Splicing 40

t. RNA Splicing endonuclease ����� 2 ������� intron Ø Splicing ligase ����� 2 exons

t. RNA Splicing endonuclease ����� 2 ������� intron Ø Splicing ligase ����� 2 exons ������ Ø 41

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Reverse transcription ����� DNA ������ RNA ����� ��� ��� RNA-dependent DNA polymerase ���� Reverse

Reverse transcription ����� DNA ������ RNA ����� ��� Ø��� RNA-dependent DNA polymerase ���� Reverse transcriptase (RT) ØDNA ������� Complementary DNA (c. DNA) ØRNA tumor virus ��������� c. DNA ���������� (integrate) ���� DNA ��� host Ø 43

Reverse Transcription 44

Reverse Transcription 44

Mechanism ��� Reverse Transcription 45

Mechanism ��� Reverse Transcription 45

Central Dogma Update V. 1 V. 2 V. 3 V. 4 )forbidden( 46

Central Dogma Update V. 1 V. 2 V. 3 V. 4 )forbidden( 46