Signals in DNA tell RNA polymerase where to start and stop transcription

1. Signals in DNA tell RNA polymerase where to start and stop transcription • Synthesis starts at a promoter, a conserved sequence 5’ of a gene. • Chain elongation occurs until RNA polymerase encounters a termination sequence on the DNA. • RNA polymerase releases the single stranded RNA and the double stranded DNA template upon encountering a termination sequence. • This slide depicts a bacterial RNA polymerase. Transcription initiation in eukaryotic cells is more complicated.

2. Promoter and terminator sequences in bacteria

3. Clicker question Propose a mechanism by which the highlighted areas could cause transcription to stop. 5) The first two sequences are inverted repeats of each other, thus a hairpin, which is a signal for termination.

4. What is the same in all* somatic cells of your body? DNA RNA Proteins Lipids Carbohydrates Lysosomes * Some immune cells are interesting exceptions.

5. The DNA in differentiated cells contains all the instructions to make a new organism This is reproductive cloning -- how Dolly the Sheep was created. In theory, this procedure could be done in humans, but it would be unethical because most of the embryos are abnormal. Cell types differentiate during development of a multicellular organism to become specialized (e.g., muscle, nerve, brain, blood cells are different from each other), but they contain the same DNA. What is different is that they express different genes, so they accumulate different sets of RNA and protein.

6. Cells regulate expression of proteins at many levels We will discuss transcriptional control.

7. Transcription is controlled by proteins binding to regulatory sequences on DNA Almost all prokaryotic and eukaryotic genes have regulatory DNA sequences that are used to switch the gene on or off. Gene regulatory proteins bind to the regulatory DNA sequences. - Repressors prevent transcription - Activators promote transcription Early studies of gene regulation involved bacteriophage and bacterial genes, which have simpler regulatory regions than eukaryotic genes.

8. Repressors block RNA polymerase -- prevent transcription initiation Activators interact with RNA polymerase to help initiate transcription

9. Bacteriophages (bacteria eaters) --Viruses that infect bacteria After infection, phage can live in one of two states Lytic: phage degrades host DNA, hijacks host cell machinery to produce many new phage. When cell is depleted, phage lyse their host bacterium to release new phage. Lysogenic: phage DNA integrates into host chromosome. Prophage (phage DNA) duplicated every time host divides. Stress to host causes excision of prophage DNA and entry into lytic phase. Classic example of transcriptional regulation to turn on and off genes

10. Viruses that infect bacteria (bacteriophage or phage) look like syringes Levine, A. “Viruses” p. 34

11. Bacteriophage infection www.seyet.com/t4_academic.html

12. Growth of phage  Figure 1.2. Mark Ptashne. A Genetic Switch: Gene Control and Phage 

13. (cI protein also called  repressor) Genetic switch between two states in phage  Lysogenic state

14. Lysogenic stateLysogenic genes on; lytic genes off repressor (cI protein) blocks transcription of Cro (a lytic gene) and activates transcription of its own gene.  Repressor gene is on Phage genes are off A repressor physically blocks RNA pol from initiating transcription. An activator interacts with RNA pol to promote transcription. ”repressor” acts as both a repressor and an activator

15. Lytic stateLytic genes on; lysogenic genes off Cro repressor binds to OR3, preventing transcription of  repressor (cI protein) and other lysogenic genes. RNA pol can now transcribe Cro gene.  Repressor gene is off Phage genes are on

16. Repressor gene is off Phage genes are on Lytic state: UV light or other inducer causes phage genes to be turned on and new phage to be produced, which results in lysis of the host cell. Viral repressors (green spheres) prevent transcription of lysogenic genes, but not lytic genes. A genetic switch in prokaryotes (lysis versus lysogeny of a virus) is controlled by regulation of transcription Repressor gene is on Phage genes are off Lysogenic state: Bacteriophage genome is integrated into bacterial genome. Bacteriophage is dormant and phage genes are not expressed. Viral repressor proteins (red dumbbells) block transcription of lytic genes and activate transcription of repressor genes.

17. Transcriptional regulators (repressors, activators) are proteins that recognize specific DNA sequences • a-helices of proteins fit into the major groove of DNA. • Amino acid sidechains from the protein make specific contacts with exposed edges of basepairs. Structure of bacteriophage lambda repressor Beamer & Pabo, 1992, J. Mol. Biol. 227: 177.

18. Sequence-specific recognition of DNA by proteinsSeeman et al. (1976) PNAS 73:804-808 Major groove Minor groove Major groove A A D A D A Minor groove

19. Clicker question Many DNA binding proteins (including restriction enzymes) recognize palindromic* sequences of DNA. What does binding to a palindromic DNA sequence imply about the structure of a DNA-binding protein? 1) It is a dimer with translational symmetry It is a dimer with two-fold rotational symmetry It is a dimer with mirror (inversion) symmetry 4) It is a trimer with translational symmetry It is a trimer with three-fold rotational symmetry It is a trimer with three-fold mirror symmetry GAAGCTCGTACGAGCTTC CTTCGAGCATGCTCGAAG *Palindrome examples: MADAM I’M ADAM or

20. Clicker question What does binding to a palindromic DNA sequence imply about the structure of the DNA-binding protein? 1) It is a dimer with translational symmetry 2) It is a dimer with two-fold rotational symmetry 3) It is a dimer with two-fold mirror symmetry GAAGCTCGTACGAGCTTC CTTCGAGCATGCTCGAAG *Palindrome examples: MADAM I’M ADAM or

21. Transcriptional regulation of eukaryotic genes is more complicated than regulation of prokaryotic genes • Basal promoter elements (e.g., TATA box: 25 bp upstream of transcription start site). • Promoter proximal element. 100-200 bp long -- close to site of transcription initiation. Contains sequences recognized by different transcription factors. • Enhancer elements. Can be a few thousand to 20,000 bp upstream or downstream from the initiator site.

22. TBP Transcriptional activation involves interactions over long stretches of DNA • TATA box binding protein (TBP) binds to RNA polymerase and other proteins to form pre-initiation complex.

23. TATA-binding protein (TBP)

24. Transcriptional activation involves interactions over long stretches of DNA • TATA box binding protein (TBP) binds to RNA pol and other proteins to form pre-initiation complex. • Pre-initiation complex interacts with different specific transcription factors bound to promoter proximal elements and enhancer elements. • Each gene in every cell has same DNA control sequences, but not every cell has complete set of DNA binding proteins to turn on every gene.

25. NFkB Stress signal comes from outside the cell. • NFkB proteins are cytoplasmic. Inactive when bound to I-kB. • Inducers (stress, infection, etc.) trigger dissociation and degradation of I-kB, then NFkB enters nucleus and activates genes. • NFkB binds to kB sites in the enhancer regions of genes involved in cellular defense mechanisms and differentiation. • NFkB induces transcription of HIV viral RNA by the host cell RNA polymerase. • David Baltimore’s lab at Caltech works on NFkB. A signaling cascade in the cytoplasm results in degradation of I-kB. NFkB enters the nucleus and activates genes.

26. Crystal structure a piece of NFkB bound to a kB site on DNA • Structure resembles butterfly with protein domains as wings attached to cylindrical body of DNA. • Contacts with DNA formed by loops between b-strands. No helical or sheet structure at recognition surface. Ghosh et al., 1995, Nature 373: 303-310; Müller et al., ibid, 311-317

27. How can you tell when/where a gene is being expressed?

28. Hybridization can be used to locate when/where a gene is expressed or compare genomes • Take advantage of ability of DNA to pair selectively with a second strand of complementary nucleotide sequence. • Denature DNA (heat or pH) to separate strands, then slowly reverse to allow double helices to reform (hybridization or renaturation). • Any two complementary single-stranded nucleic acid chains can hybridize (DNA/DNA, RNA/RNA, RNA/DNA). [HIV reverse transcriptase makes cDNA, or complementary DNA, from a single-stranded RNA template.] Figure 10-12, Little Alberts

29. Physical Chemistry of DNA Hybridization Studied at Caltech in 60s and 70s by Norman Davidson 1. The hydrogen bonds that form double-stranded DNA are easily disrupted by heating. 2. Some dyes fluoresce when they bind to double-stranded DNA.

30. 2 mm Can use hybridization to search for gene expression • Make a ssDNA probe (can be synthesized) to detect complementary sequences of interest. • Can probe for normal versus mutant forms of a gene (e.g., sickle-cell anemia gene; cancer-predisposing genes). • Can probe cDNA from cells at a particular developmental stage or from a tissue to see when or where a gene is expressed. • Can do hybridizations in situ (in place) to locate nucleic acid sequences in cells, organisms (below left) or on chromosomes (below right). FISH -- Fluorescence In Situ Hybridization Whole mount zebrafish in situ hybrization kit

31. DNA microarrays to evaluate gene expression Array thousands of DNA oligonucleotides (probes) in known locations, each is a different sequence Add target cDNA (complementary DNA made from transcribed RNA) (target) to hybridize under high-stringency conditions Probe-target hybridization detected by and quantified by fluorescence

32. Commercial Gene Chips Probes attached covalently to a chemical matrix on a solid surface (quartz). Made using photolithography.

33. Applications Gene expression profiling -- monitor mRNA for thousands of genes to study effects of: different stages in development or differentiation disease (cancer) infection Compare genomes in different organisms Identify single nucleotide polymorphisms (SNPs) for genotyping, forensics, to study predisposition to disease, find mutations in cancers Alternative splicing detection -- use probes for predicted exons of a gene

34. Red Fluorescent ProbesGreen Fluorescent Probes Dual Color Microarray experiment n.m.wikipedia.org/wiki/DNA_microarray

35. ~40,000 probe spotted oligo microarray Yellow -- merge of red and green fluorescence n.m.wikipedia.org/wiki/DNA_microarray