1. Promoters Map ends of mRNA on DNA Mapping sites on DNA for protein binding General Properties of promoters Bacterial Promoters Promoters for eukaryotic RNA polymerases

2. Nuclease Protection : Map the nucleotide in DNA that encodes the 5’ end of mRNA. 5’ 3’ nontemplate template RNA Hybridize RNA with a DNA probe labeled on 5’ end: S1 nuclease (single strand specific) Denaturing gel electrophoresis Size = distance from labeled site to first discontinuity between DNA and RNA, e.g. 5’ end of gene or beginning of an exon.

3. Nuclease Protection : Map the nucleotide in DNA that encodes the 3’ end of mRNA. 5’ 3’ template RNA Hybridize RNA with a DNA probe labeled on 3’ end: S1 nuclease (single strand specific) Denaturing gel electrophoresis Size = distance from labeled site to first discontinuity between DNA and RNA, e.g. 3’ end of gene or end of an exon. nontemplate

4. Primer Extension : Another method to determine DNA sequence encoding the 5’ end of mRNA 5’ 3’ nontemplate template RNA Reverse transcriptase + dNTPs to extend primer to 5’ end of RNA Denaturing gel electrophoresis Size = distance from labeled site to the 5’ end of the mRNA Anneal a primer, complementary to RNA, labeled on 5’ end:

5. Rapid amplification of 5’ cDNA ends = 5’ RACE 5’ 3’ RNA cDNA CCCCC GGGG Reverse transcriptaseCopies RNA to end, adds 3-5 C’s Anneal oligo-nt with G’ s on 3’ end CCCCC cDNA GGGG CCCCC “RACE-ready cDNA” GGGG cDNA CCCCC GGGGG 5’ Further extension by RT’ase of oligo-nt template Primers, Taq polymerase, dNTPs 25-35 cycles to amplify 5’ end of cDNA by PCR

6. Methods for identifying the sites in DNA to which proteins bind in a sequence- specific manner Electrophoretic Mobility Shift Assays DNA Footprinting Analysis DNase Protection Exonuclease Protection Methylation Interference

7. Does a protein bind to a particular region? (Electrophoretic Mobility Shift Assay) A short DNA fragment will move relatively fast in a non-denaturing polyacrylamide gel. A DNA fragment bound to a protein will move much more slowly. The mobility of the protein is the primary determinant of the mobility of the protein-DNA complex. Different protein-DNA complexes usually migrate at different rates. Can test for sequence-specificity by adding increasing amounts of competitor DNAs

8. Example of EMSA The radioactively-labeled DNA probe binds two proteins. Sp1 (or something sharing its binding site) is in complex A. Cannot determine the identity of protein in complex B from these data. Each is sequence specific.

9. To what specific sequence in DNA does the protein bind ? (DNA Footprinting Assay) A protein bound to a specific sequence within a DNA fragment will protect that sequence from cleavage by DNase or chemical reagents. DNA outside the region of protein binding will be sensitive to cleavage. After cleavage and removal of the protein, the resulting fragments of labeled DNA are resolved on a denaturing polyacrylamide gel. Protein-protected DNA results in a region with no bands on the gel (a “footprint”); the distance from the labeled site is determined by flanking bands.

10. DNase footprint assay, part 1 Protein bound specifically to DNA protects the DNA from cleavage by DNase at discrete sites.

11. DNase footprint assay, Part 2 Specific locations of protected segments show the binding site(s) for the protein.

12. Example of DNase footprint analysis: DctA bound to DNA Purified DctA binds to two sites on DNA. Data from Dr. Tracy Nixon.

13. Methylation Interference Assay determines the nucleotides required for binding a protein

14. General Properties of Promoters

15. General features of promoters A promoter is the DNA sequence required for correct initiation of transcription It affects the amount of product from a gene, but does not affect the structure of the product. Most promoters are at the 5’ end of the gene.

16. Phenotypes of promoter mutants Promoters act in cis, i.e. they affect the expression of a gene on the same chromosome. Let p = promoter; lacZ is the gene encoding beta-galactosidase. p + lacZ - /p + lacZ + : Phenotype is Lac +, i.e. lacZ + complements lacZ ‑ in trans. p + lacZ - /p - lacZ + : Phenotype is Lac -, i.e. p + does not complement p ‑ in trans.

17. Promoter alleles show cis-dominance The allele of the promoter that is in cis to the active reporter gene is dominant. A wild-type promoter will drive expression of a wild-type gene, but a defective promoter will not drive expression of an wild-type gene. p + lacZ - /p - lacZ + : Phenotype is Lac -, i.e. p - cannot drive expression of lacZ +. p + lacZ + /p - lacZ - : Phenotype is Lac +, i.e. p + can drive expression of lacZ +.

18. Bacterial promoters A combination of approaches shows that the -10 TATAAT and -35 TTGACA sequences are the essential DNA sequences in most E. coli promoters –Conservation of DNA sequences 5’ to genes –Sequence of mutations that increase or decrease the level of accurate transcription –DNA sequences contacted by RNA polymerase –Region protected from nucleases by binding of RNA polymerase is -50 to +20.

19. -35 and -10 sequences ---TTGACA-----------TATAAT-----CAT--- ---AACTGT-----------ATATTA-----GTA--- -3516-19 bp-10+14-8 bp Unwound in open complex Promoter mutants Contacts with RNA polymerase The sigma subunit of RNA polymerase contacts both the -35 and the -10 boxes.

20. Alternate  factors are used to express specific sets of genes FactorGeneUse -35Separation -10 General Heat Shock Flagella Nitrogen starvation         rpoD rpoH fliA rpoN TTGACA CCCTTGAA CTAAA CTGGNA 16 - 19 bp 13 - 15 bp 15 bp 6 bp TATAAT CCCGATNT GCCGATAA TTGCA

21. Promoters for eukaryotic RNA polymerases

22. Use of site-directed mutagenesis to define the promoter Use site-directed mutations (deletions and point mutations) in the DNA sequence to test promoter activity. Ligate the mutated DNA fragments to the coding region of a reporter gene. –Any gene: assay for stable RNA whose 5’ end is at the start site for transcription. –beta-galactosidase: measure the hydrolysis of an analog of lactose that generates a colored, fluorescent or chemiluminescent product –Luciferase: chemiluminescent reaction

23. Tests for expression of the target gene Whole organisms –Transgenic animals, plants or other species in which the DNA construct has been integrated into the genome Cell lines –Transfected (transformed) cells (animal, plant, fungal, bacterial) carrying the DNA construct either unintegrated or integrated into the genome Cell-free systems –Crude cell extracts –Purified RNA polymerase plus GTFs plus other desired proteins

24. Example of mutational analysis of a eukaryotic promoter, Fig. 3.2.8 Decrease in RNA produced if TATA box is deleted. It is needed for transcription both in vivo and in vitro. Decrease in RNA produced if CCAAT box is deleted. It is needed for transcription in vivo. “Linker scanning” mutations (clusters of point mutations) reveal specific short sequences needed for transcription.

25. Evidence for a Pol II promoter Conserved in many “Class II” genes Conserved in mammalian HBB genes HBB, encodes beta-globin Directed mutation: loss of trans- cription Mutations cause beta-thalassemia Specific binding of transcription factors Mutation of gene encoding transcription factor that binds here prevents HBB expression

26. Promoter for RNA Polymerase II Regulate efficiency of utilization of minimal promoter Minimal promoter: TATA + Inr

27. Minimal promoter is needed for basal activity and accurate initiation Minimal promoter is needed for the assembly of the initiation complex at the correct site. TATA box –Well-conserved sequence centered about 25 bp 5’ to start site –TBP and TFIID bind Initiator –Short segment around start site: YANWYY, where A is the start site Y = T or C, W = T or A –Part of TFIID will bind here

28. Additional sequences, usually upstream, regulate the amount of expression Binding sites for transcriptional regulatory proteins are often found upstream of the minimal promoter. Binding of transcriptional activators will increase the amount of transcription from the promoter –Sp1 binds GGGGCGGGG –CP1 binds CCAAT Binding and/or effects of activators can be regulated,e.g. in response to hormones and other signals. Repressors and silencing proteins decrease the amount of transcription.

29. Promoter for RNA polymerase I

30. Promoter for RNA polymerase III

31. Enhancers : Additional DNA sequences that regulate transcription Enhancers cause an increase in expression of a gene. Can act in either orientation. Can act in a variety of positions: –5’ to gene (similar to an upstream activation sequence) –Internal to a gene (e.g. in an intron) –3’ to a gene Can act at a considerable distance from the gene (up to at least 50 kb in some cases). Contain a set of binding sites for transcriptional activators.

32. Where is the 5’ end of BMB6? Hybridize RNA with DNA probes labeled on 5’ end: S1 nuclease (single strand specific) Denaturing gel electrophoresis 0 500100015002200 600 bp500 bp Hybridize to RNA 600 bp probe: 100 nt protected, labeled fragment 500 bp probe: NO protected, labeled fragment

33. Where is the 5’ end of BMB6? Answer Hybridize RNA with DNA probes labeled on 5’ end: S1 nuclease (single strand specific) Denaturing gel electrophoresis 0 500100015002200 600 bp500 bp Hybridize to RNA 100 nt protected, labeled fragmentNO protected, labeled fragment 1600

34. Where is the promoter for BMB6? 0 500100015002200 1600 2200 Luciferase ABCD activity 100 50 Luciferase 0

35. Where is the promoter for BMB6? Answer 0 500100015002200 1600 2200 Luciferase ABCD activity 100 50 Luciferase 0 50 0 0