1. Chapter 11: Transcription Initiation Complex Copyright © Garland Science 2007

2. Genome expression includes 2 steps Initiation of transcription. Assembly of upstream protein complex (e.g. RNA polymerase & accessory proteins) This step determines whether a gene should be expressed or not. Synthesis & processing of RNA (next Chapter). RNA polymerase synthesizes mRNA & subsequently processes or modifies into mature mRNA.

3. Figure 11.1 Genomes 3 (© Garland Science 2007)

4. Table 11.1 Genomes 3 (© Garland Science 2007) 11-1. DNA binding proteins are the key to initiate transcription. DNA binding proteins play a wide variety of functions (e.g. RNA transcription, DNA replication, repair, recombination, etc.)

5. Figure 11.2 Genomes 3 (© Garland Science 2007) 11-1-1. (Cont.) Proteins contain highly specific regions in direct contact w/DNA (called “DNA binding motifs”). Helix-turn-helix motif (20 AA in length) common in both pro- & eukaryotes; consists of two α-helix units & one β-turn; 2nd α- helix recognizes/contacts DNA major groove; in bacteria: lactose repressor; in eukaryotes: POU domain & winged HTH motif.

6. Figure 11.4 Genomes 3 (© Garland Science 2007) 11-1-1. (Cont.) Zinc fingers common in eukaryotes; 1% mammalian genes encode zinc fingers; 6 types; well-studied Cys 2 His 2 finger consists of one α- helix units & one β-sheet; α-helix recognizes/contacts DNA major groove; Zinc atom to stabilize the finger structure; a single protein sometimes contains multiple copies of zinc fingers.

7. Figure 11.6 Genomes 3 (© Garland Science 2007) 11-1-1. (Cont.) Ribbon-helix-helix motif common in bacteria; the ribbon (β- sheets) contacts DNA major groove. TATA binding protein (or TBP domain) contacts minor groove of DNA. RNA binding proteins include RNP domain, dsRNA binding domain, κ- homology domain, etc.

8. Figure 11.7 Genomes 3 (© Garland Science 2007) 11-1-2. DNA-binding sites in a genome Attachment sites for DNA-binding proteins are usually located immediately upstream of a gene; help to identify real genes in a genome (e.g. to search several Kb upstream).

9. Figure 11.8 Genomes 3 (© Garland Science 2007) 11-1-2. (Cont.) Identification of DNA binding protein by experimental techniques Gel retardation Restriction collection mixed with nuclear proteins; DNA-protein complex impede gel electrophoresis.

10. Figure 11.9 Genomes 3 (© Garland Science 2007) 11-1-2. (Cont.) Modification protection assay 1 Restriction fragments end labeled; mixed with nuclear proteins; add nuclease under limiting conditions to make 1 random cut per fragment; DNA- protein complex will not be digested; compare gel electrophoresis.

11. Figure 11.10 Genomes 3 (© Garland Science 2007) 11-1-2. (Cont.) Modification protection assay 2 Restriction fragments end labeled; mixed with nuclear proteins; add DMS under limiting conditions to methylate 1 guanine per fragment; DNA- protein complex will not be methylated; compare gel electrophoresis.

12. Figure 11.12 Genomes 3 (© Garland Science 2007) 11-1-3. DNA sequence influence DNA binding protein Configuration effect B-form or Z-form: major groove can be “direct readout”. A-form is difficult to read. DNA bending repeated adenines cause bending at the 3’ end.

13. Figure 11.13 Genomes 3 (© Garland Science 2007) 11-1-4. Interaction between DNA & DNA binding protein Most are electrostatic & non- covalent (between - charges of DNA & + charges of protein R groups) Recognize specific (thermodynamically favorable) DNA sequences but can also bind non-specifically; dimers structure maximizes interaction w/major groove.

14. Table 11.3 Genomes 3 (© Garland Science 2007) 11-2. DNA-protein interactions to initiate transcription. 11-2-1. DNA-dependent RNA polymerase In eukaryotes: 3 distinct types (I for rRNAs, II for proteins, III for tRNAs) consisting of 8-12 subunits In bacteria: RNA polymerase consists of α 2 ββ’σ subunits

15. Figure 11.14 Genomes 3 (© Garland Science 2007) 11-2-1. (Cont.) In bacteria: RNA polymerase directly attach promoters (where RNA polymerase binds upstream of genes) In eukaryotes: DNA- binding proteins first bind & then RNA polymerase binds

16. Figure 11.15 Genomes 3 (© Garland Science 2007) 11-2-1. (Cont.) E. coli promoter contains two 6-nt segments: -35 box 5’-TTGACA-3’ -10 box 5’-TATAAT-3’ +1 is where transcription begins 20-600 nt upstream of the start codon

17. Table 11.4 Genomes 3 (© Garland Science 2007) 11-2-1. (Cont.) E. coli promoter position is relatively conserved; -35 & -10 box sequences can vary from genes to genes (see below); but mutation in promoter regions prevents gene expression.

18. Figure 11.16 Genomes 3 (© Garland Science 2007) 11-2-1. (Cont.) Eukaryotic promoter is where initiation complex is assembled; usually consists of Core promoter + upstream promoter elements. e.g. RNA polymerase II has a core promoter (TATA box + initiator sequence) plus downstream promoter element, GC- rich motif, proximal sequence element.

19. Figure 11.17 Genomes 3 (© Garland Science 2007) 11-2-2. Assembly of transcription initiation complex General steps: 1. Attach to promoter sequences; 2. Convert from a closed complex to an open complex; 3. Move away from promoter & initiate transcription.

20. Figure 11.18 Genomes 3 (© Garland Science 2007) 11-2-2. (Cont.) In E. coli: Attach to promoter sequences is specified by σ subunit & -35 box; Convert from a closed complex to an open complex is based on -10 box; σ subunit dissociates soon after transcription initiates.

21. Figure 11.19 Genomes 3 (© Garland Science 2007) 11-2-2. (Cont.) In eukaryotes: The process is similar but RNA polymerase II does not directly recognize promoter sequences; instead, general transcription factor (GTF) binds to DNA; “saddle- like” structure.

22. Figure 11.22 Genomes 3 (© Garland Science 2007) 11-3. Regulation of transcription initiation Primary regulation occurs at the level of transcription initiation & decides which gene is expressed in a particular cell & relative rate Secondary regulation is during the post- transcription (e.g. mRNA modification) and the protein synthesis & modification.

23. Figure 11.23 Genomes 3 (© Garland Science 2007) 11-3. (Cont.) Two levels of regulation: Constitutive control by promoter structure (basal level of transcription) Regulatory control by regulatory proteins (transcription initiation). In E. coli, -35 box influences σ subunit recognition & RNA polymerase attachment; strong promoters direct x1000 more productive initiations than weak promoters.

24. Figure 11.24 Genomes 3 (© Garland Science 2007) 11-3. (Cont.) Regulatory control in E. coli, the concept of operator (a region between promoter and operon & regulates the initiation of operon). A few transcripts 5000 transcripts

25. Figure 11.25 Genomes 3 (© Garland Science 2007) 11-3. (Cont.) Regulatory control in E. coli, tryptophan (the gene product itself) is a co-repressor to inactivate operon expression. The process is called “feedback inhibition”.

26. Figure 11.26 Genomes 3 (© Garland Science 2007) 11-3. (Cont.) In addition to repressors, there are activators which increase the efficiency of transcription initiation; Same proteins bind more than 1 promoters (see left); Recognition sequences can be enhancers or silencers by conformational changes.

27. Figure 11.27 Genomes 3 (© Garland Science 2007) 11-3. Regulation of transcription initiation in eukaryotes RNA polymerase II promoter consists of many short sequence regions, including core promoter (TATA box & Inr sequence), basal promoter elements (CAAT box, GC box, etc), response modules, cell- specific modules, developmental regulators.

28. Figure 11.28 Genomes 3 (© Garland Science 2007) 11-3. Regulation of transcription initiation in eukaryotes (Cont.) Alternative promoters also contribute to the transcription regulation, e.g. human dystrophin gene (the largest gene spanning 2.4 Mb w/78 introns) has >7 tissue-specific alternative promoters (e.g. cortical tissue, muscles, cerebellum, etc.)

29. Figure 11.29 Genomes 3 (© Garland Science 2007) 11-3. Regulation of transcription initiation in eukaryotes (Cont.) Activators & co-activators bind to upstream promoter elements & enhancers (activation domain); interact with RNA polymerase II & regulate a single gene or multiple genes.

30. Figure 11.30 Genomes 3 (© Garland Science 2007) 11-3. (Cont.) Activators & co-activators interact with RNA polymerase II via another protein complex called mediator. (Left) yeast mediator, detailed mechanism is not clear. Repressors are also important in eukaryotes, e.g. inhibit assembly of pre- initiation complex; activators & repressors themselves are controlled by synthesis & conformational changes.

31. Chapter 11 Summary DNA-binding proteins play a central role in transcription; many can attach to specific DNA sequences (e.g. helix-turn-helix or zinc finger); some can directly read DNA sequences in major grooves which can be affected by DNA conformation. Promoters specify where transcription initiation complex should be assembled; bacteria have a single RNA polymerase which directly attach to 2 promoter regions; eukaryotes have 3 RNA polymerases & more complex promoters which interact via general transcription factors; activators & repressors can further regulate transcription initiation.