Lecture 21 Regulation of transcription

Transcription

1 Lecture 21 Regulation of transcription Key learning goals: Understand what a promoter sequence is Understand the role of sigma factors in prokaryotic RNAP initiation Understand what an open complex is, and its importance for RNAP initiation Understand the major duties of the three eukaryotic RNAPs Know the RNAP II general transcription factors (GTFs) Define what a promoter sequence is, and what it does Define what a repressor sequence is, and what it does Understand that transcription activators bind to specific enhancer sequences to turn on specific genes, in specific cells, at specific times. Understand that transcription activators have (at least) two possible modes of action: they can bind to the mediator complex, or (next lecture) they can send signals to proteins tha modify chromatin structure.

2 Three (3) eukaryotic DNA-dependent RNA polymerases DNA RNAP I RNAP II RNAP III rrna mrna ribosome trna protein

3 RNAP catalytic subunit architecture is evolutionarily conserved 2 Mg 2+ in active site

4 Cis-acting DNA sequences called promoters tell the transcription machinery where to initiate Prokaryotes Eukaryotes (RNAP II)

5 Sigma factor (σ) proteins direct bacterial RNAP to promoters open complex formation 3. 4.

6 RNAP s cannot find promoter sequences by themselves! Prokaryotes Eukaryotes (RNAP II)

7 From here on, we ll focus on transcription in eukaryotes, not bacteria!

8 RNAP s cannot find promoter sequences by themselves! (= coding strand)

9 The following is a general outline of how RNAP II transcription is intiated. Somewhat different events occur at different promoters and in different cells, so there are many variations on the basic outline that we ll learn about in this course!

10 RNAP II and the GTFs together form the pre-initiation complex (PIC) GTFs = General Transcription Factors Roger Kornberg, Nobel Lecture

11 RNAP II and the GTFs together form the pre-initiation complex (PIC) Roger Kornberg, Nobel Lecture

12 TFII = general Transcription Factor for Pol II Mechanism of initiation of RNA polymerase II transcription H H Pol F Pol BN Factor Role BBC C TBP TBP TFIIB TFIIE TFIIF TFIIH Configure DNA to the pol II surface Direct DNA path, stabilize early transcribing complex Recognize closed complex, recruit TFIIH Capture nontemplate strand upon melting Introduce negative superhelicity in promoter DNA Roger Kornberg, Nobel Lecture

13 PIC: Pre-Initiation Complex: General Transcription Factors (GTFs) & RNAP II assemble at a eukaryotic promoter TATA-binding protein (TBP) is a key subunit of TFIID TBP introduces a 45 bend into the double helix, locally untwisting the DNA TBP is a universal GTF, required for RNAP I, II, and III initiation

14 Path of the DNA template in the PIC H F TBP Roger Kornberg, Nobel Lecture

15 Initiation: formation of open complex (transcription bubble) When the DNA melts, TFIIF captures the non-template (coding) strand

16 Enhancer sequences regulate the activity of core promoter Prokaryotes Eukaryotes (RNAP II) Enhancers can be hundreds or even tens of thousands of bp distant from the promoter!

17 Enhancer sequences regulate the activity of core promoter Specific enhancer sequences bind different activator transcription factors Each gene is controlled by a different array of enhancer sequences

18 Sets of enhancer sequences bind different combinations of activators to give modular control over each gene s promoter

19 Enhancer sequences regulate the activity of core promoter enhancer sequence activator protein GTFs? RNAP II TATA promoter sequence

20 Many activators regulate RNAP II by interacting with mediator, a huge protein complex of >20 subunits enhancer sequence GTFs x activator protein RNAP II TATA promoter sequence enhancer sequence GTFs activator protein mediator complex Conserved Mediator subunits yeast Drosophila human Med1 Trap220* TRAP220 Med4 Trap36 TRAP36 Med6 Med6 hmed6 Med7 Med7* hmed7 Med8 Arc32* hnut2 Cse2/Med9 CG5134* MED9 Nut2/Med10 Nut2* TRAP230 Med11 Med21 MED11 Srb8 Kto* TRAP240 Ssn2/Srb9 Skd/Pap/Bli* TRAP170 Rgr1 Trap170 TRAP95 Gal11 Arc105* MED15 Sin4 Trap95* TRAP80 Srb4 Trap80 htrfp Srb5 p28/cg14802 MED18 Rox3 CG5546* MED19 Srb2 Trfp hsrb7 Srb7 Trap19 TRAP150b Srb6 Med24 MED22 Med1 Trap220* TRAP220 CDK8 cycc CDK8 cycc CDK8 cycc RNAP II TATA promoter sequence Mediator is required for efficient activated transcription from all RNAP II promoters

21 Sets of enhancer sequences bind different combinations of activators to give modular control over each gene s promoter