1. Lecture 8 Transcription Initiation Prokaryotic Eukaryotic Reading: Chapter 4 (115-116) Chapter 11 Molecular Biology syllabus web siteweb site

2. Bacterial transcription initiation RNA polymerase initiates transcription of most genes at a unique DNA position lying upstream of the coding sequence The base pair where transcription initiates is termed the transcription-initiation site or start site By convention, the transcription-initiation site in the DNA sequence is designated +1, and base pairs extending in the direction of transcription (downstream) are assigned positive numbers which those extending in the opposite direction (upstream) are assigned negative numbers Various proteins (RNA polymerase, activators, repressors) interact with DNA at or near the promoter to regulate transcription initiation

3. Differences in E. coli promoter sequences affect the frequency of transcription initiation

4. Lac Operon Beta Galactosidase (lacZ) Permease (lacY) Transacetylase (lacA) lacZ lacY lacALac promoter mRNA protein

5. LacZ ( -galactosidase)

9. Eukaryotes

10. Promoter deletions to identify transcriptional control sequences

15. Run-off transcription: to map transcription initiation sites METHOD 1.Isolate nuclei from specific tissues 2.Incubate: nuclear extract, restriction fragments containing start site, 32P-UTP 3. Synthesized RNA is separated by electrophoresis

16. Common Eukaryotic Promoter Consensus: TATA box Alternate promoters: initiators- degenerate consensus GC-rich region (giving rise to multiple start sites)

17. Run-on transcription assay rate of transcription initiation METHOD 1. Isolate nuclei from specific tissues 2. Label growing RNA chains with 32P-UTP 3. Hybridize to filters containing test cDNAs

18. S1 nuclease mRNA 32P-labelled DNA probe mRNA 32P-labelled DNA probe denature gel electrophoresis S1 protection to assay transcription initiation rates

19. Enhancer elements cis-acting sequences that serve to increase rate of transcription initiation regardless of their location near the gene (first discovered in SV40 viral genome). S1 Protection Assay: to test rate of transcription initiation METHOD 1. Transfect construct with/without enhancer into test cells 2. Isolate RNA and hybridize with 32P-labeled DNA probe 3. Treat with S1 nuclease, denature, subject to gel electrophoresis

20. Typical eukaryotic gene

21. Column purified fractions of DNA-binding proteins incubated with test promoter fragment Gel-shift assays identify general region of protein-DNA interaction METHOD 1. Isolate nuclei and prepare protein extract or purify DNA binding proteins 2. Incubate with restriction fragment or short DNA 3. Separate by agarose gel electrophoresis Controls: non-specific DNA; no extract

22. METHOD 1. Isolate restriction fragment 2. Add DNA-binding protein 3. Partially digest with DNaseI 4. Separate by polyacrylamide gel electrophoresis Controls: no protein The footprint of RNA polymerase and lac repressor on the lac control region DNase I footprinting assays identify specific regions of protein-DNA interactions

23. The lac control region contains three critical cis-acting sites Figure 10-9

24. How to demonstrate the specificity of a putative transcription factor? In vitro In vivo

25. In vitro METHOD 1. Set up in vitro transcription reaction with DNA template, 32P-UTP, plus or minus transcription factor 2. Separate transcripts by gel electrophoresis Controls: non-specific DNA; no protein

26. In vivo METHOD 1.Prepare two constructs: a) encoding transcription factor X b) reporter gene downstream of transcription factor binding site plus minimal promoter 2. Transfect cells and assay for reporter gene activity Controls: transfect each plasmid alone

27. Transcription factors have separate domains DNA binding Transcription activation

28. Yeast Two-hybrid SystemYeast Two-hybrid System- to fish out interacting transcription factors