《分子生物学 Molecular Biology》课程教学课件（PPT讲稿，英文版）Chapter 10 Eukaryotic RNA Polymerases and Their Promoters

Chapter 10 Eukaryotic RNA Polymerases and Their Promoters Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display. David A.Bushnell,Kenneth D.Westover,and Roger D.Komberg

Chapter 10 Eukaryotic RNA Polymerases and Their Promoters

Prokaryotes have only one RNA polymerase that makes three kinds of RNAs (mRNA,rRNA,and tRNA)(switch o-factor,the core enzyme is same) What is the situation in eukaryotes? 10-2

10-2 Prokaryotes have only one RNA polymerase that makes three kinds of RNAs (mRNA, rRNA, and tRNA)（ switch σ-factor，the core enzyme is same) What is the situation in eukaryotes?

10.1 Multiple Forms of Eukaryotic RNA Polymerase Ribosomal genes are different from other nuclear genes GC content(60%),compared with the rest of the DNA(40%) Unusually repetitive Nucleolus (is the site to synthesize rRNAs There are at least two RNA polymerases operating in eukaryotic nuclei One transcribes major ribosomal RNA genes One or more to transcribe rest of nuclear genes 10-3

10-3 10.1 Multiple Forms of Eukaryotic RNA Polymerase ✓ Ribosomal genes are different from other nuclear genes – GC content (60%), compared with the rest of the DNA (40%) – Unusually repetitive – Nucleolus (核仁) is the site to synthesize rRNAs ✓ There are at least two RNA polymerases operating in eukaryotic nuclei – One transcribes major ribosomal RNA genes – One or more to transcribe rest of nuclear genes

Three Nuclear Polymerases Eukaryotic nuclei contain three RNA polymerases -These can be separated by ion-exchange chromatography RNA polymerase I found in nucleolus -Location suggests in transcribes rRNA genes RNA polymerases ll and Ill are found in the nucleoplasm(核浆) 10-4

10-4 Three Nuclear Polymerases ✓ Eukaryotic nuclei contain three RNA polymerases – These can be separated by ion-exchange chromatography ✓ RNA polymerase I found in nucleolus – Location suggests in transcribes rRNA genes ✓ RNA polymerases II and III are found in the nucleoplasm (核浆)

Roles of the Three RNA Polymerases √Polymerase I makes Table 10.1 Roles of Eukaryotic RNA Polymerases large rRNA precursor RNA Cellular RNAs Mature RNA √Polymerase ll makes Polymerase Synthesized (Vertebrate) Large rRNA precursor 28S,18S,and -Heterogeneous 5.8S rRNAs hnRNAs mRNAs nuclear RNA(核不均一 snRNAs snRNAs 5S rRNA precursor 5S rRNA RNA,hnRNA) tRNA precursors tRNAs U6 snRNA(precursor?) U6 snRNA -Small nuclear RNA 7SL RNA (precursor?) 7SL RNA 7SK RNA (precursor?) 7SK RNA √Polymerase Ill makes precursors to tRNAs, 5S rRNA and other small RNA 10-5

10-5 Roles of the Three RNA Polymerases ✓ Polymerase I makes large rRNA precursor ✓ Polymerase II makes – Heterogeneous nuclear RNA (核不均一 RNA, hnRNA) – Small nuclear RNA ✓ Polymerase III makes precursors to tRNAs, 5S rRNA and other small RNA

RNA Polymerase Subunit Structures RPB:RNA Polymerase B(Il) CopyrightThe McGraw-Hill Companies,Inc.Permission required for reproduction or display. Table 10.2 Human and Yeast RNA Polymerase ll Subunits Resemble the RNA Yeast Protein Polymerase of prokaryotic Subunit Yeast Gene (kD) Features hRPB1 RPB1 192 Contains CTD:binds DNA:involved in start site selection:B'ortholog hRPB2 RPB2 139 Contains active site;involved in start site selection,elongation rate;B ortholog hRPB3 RPB3 35 May function with Rpb11 as ortholog of the a dimer of prokaryotic RNA polymerase hRPB4 RPB4 25 Subcomplex with Rpb7;involved in stress response hRPB5 RPB5 25 Shared with Pol ll,Ill:target for transcriptional activators hRPB6 RPB6 18 Shared with Pol l,ll,Ill;functions assembly and stability hRPB7 RPB7 19 Forms subcomplex with Rpb4 that preferentially binds during stationary phase hRPB8 RPB8 17 Shared with Pol I,ll,Ill;has oligonucleotide/oligosaccharide-binding domain hRPB9 RPB9 14 Contains zinc ribbon motif that may be involved in elongation:functions in start site selection hRPB10 RPB10 ⊙ Shared with Pol l.ll,ll hRPB11 RPB11 14 May function with Rpb3 as ortholog of the a dimer of prokaryotic RNA polymerase hRPB12 RPB12 8 Shared with Pol l,ll,ll Source:Reprinted with permission from the Annual Review of Genetics The three RNA Polymerase have several subunits in common

10-6 RNA Polymerase Subunit Structures RPB: RNA Polymerase B(II) Resemble the RNA Polymerase of prokaryotic The three RNA Polymerase have several subunits in common

12 34 Which subunits copurify with 35S 32P polymerase activity?And which Epitope + are actually subunits of the Rpb1 enzyme? Rpb2 Epitope Tagging(表位标记) phosphorylate Add an extra domain (epitope) to one subunit(RPB3),Other Rpb3 subunits normal Polymerase labeled by growing Rpb4 in labeled amino acids Rpb5- √Purify with antibody Rpb6- Denature with detergent and separate on a gel Rpb7- Rpb8 Rpb9 Rpb10

10-7 Epitope Tagging (表位标记） ✓ Add an extra domain (epitope) to one subunit (RPB3), Other subunits normal ✓ Polymerase labeled by growing in labeled amino acids ✓ Purify with antibody ✓ Denature with detergent and separate on a gel Which subunits copurify with polymerase activity? And which are actually subunits of the enzyme? phosphorylate

Polymerase Il Original 10 subunits are placed in 3 groups: Core-related in structure and function to bacterial core subunits Common-found in all 3 nuclear RNA polymerases Nonessential subunits-conditionally dispensable for enzymatic activity 10-8

10-8 Polymerase II Original 10 subunits are placed in 3 groups: ✓Core – related in structure and function to bacterial core subunits ✓Common – found in all 3 nuclear RNA polymerases ✓Nonessential subunits – conditionally dispensable for enzymatic activity

Core Subunits Three polypeptides,Rpb1,Rpb2,Rpb3 are absolutely required for enzyme activity √These are homologous to B'-,β-，anda-subunits Both Rpb1 and B'-subunit binds DNA Rpb2 and B-subunit are at or near the nucleotide- joining active site Rpb3 does not resemble a-subunit -There is one 20-amino acid subunit of great similarity 2 subunits are about same size,same stoichiometry -2 monomers per holoenzyme 10-9

10-9 Core Subunits ✓ Three polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activity ✓ These are homologous to b’-, b-, and a-subunits ✓ Both Rpb1 and b’-subunit binds DNA ✓ Rpb2 and b-subunit are at or near the nucleotide￾joining active site ✓ Rpb3 does not resemble a-subunit – There is one 20-amino acid subunit of great similarity – 2 subunits are about same size, same stoichiometry – 2 monomers per holoenzyme

Common Subunits There are five common subunits -Rpb5 -Rpb6 -Rpb8 -Rpb10 Rpb12 Little known about function They are all found in all 3 polymerases Suggests play roles fundamental in transcription 10-10

10-10 Common Subunits ✓There are five common subunits – Rpb5 – Rpb6 – Rpb8 – Rpb10 – Rpb12 ✓Little known about function ✓They are all found in all 3 polymerases ✓Suggests play roles fundamental in transcription