1. Lecture 9 Chapter 6 Gene expression and regulation II Neal Stewart

2. Focus questions How important are cis-regulatory elements and trans-acting factors in gene regulation? What are the control points that can regulate gene expression?

3. Transcription revisited

4. Promoter elements not required for transcription initiation CAAT box – usually located at -70 to -80 within the promoter GC box Other gene-specific elements (light- responsive, nutrient-responsive, etc.) Enhancer elements

5. What are some biological roles of transcription factors? Basal transcription regulation – general transcription factors Development Response to intercellular signals Response to environment Cell cycle control

6. The CRT/DRE response element responds to dehydration and cold-induced transcription factors (CBF)

7. Figure 6.7 Transcription factors

8. Figure 6.8

9. Enhancer can work from downstream and upstream region

11. Enhancers Their location is not fixed. Location could be in the upstream or downstream DNA, in intron, exon or in the untranslated region. They enhance transcription by acting on promoter in cis (typically) Each enhancer has its own binding protein. These proteins are trans-regulatory activating factors Sequence of enhancers is variable. Enhancers regulate tissue-specific and temporal expression of genes.

12. TATA binding protein (TBP) transcription factor Wikipedia.com

13. DNA-binding domains allow transcription factors to bind directly to a cis-regulatory element Helix-loop-helix Zinc finger domain Leucine zipper domain

14. Extreme trans-acting effectors of transcription: TAL effectors From plant pathogenic bacteria Xanthomonas Secreted by bacteria when they infect Transcriptional activator-like (TAL) effectors bind with plant promoters to express genes beneficial for the bacteria

15. http://www.sciencemag.org/content/333/6051/1843/F2.large.jpg

16. Repression of transcription TFs that act as repressors

17. Some trans-acting elements prevent transcription

18. Introducing RNAi http://www.youtube.com/watch?v= H5udFjWDM3E&feature=related

19. What is a microRNA (miRNA)? Controlling gene expression post-transcriptionally. microRNA is an abundant class of newly identified small non-coding regulatory RNAs. Major characteristics of miRNAs: 18-26 nt in length with a majority of 21-23 nt non-coding RNA derived from a precursor with a long nt sequence this precursor can form a stem-loop 2 nd hairpin structure the hairpin structure has low minimal free folding energy (MFE) and high MFE index Slide courtesy of Baohong Zhang, East Carolina Univ

20. miRNA regulates plant development WT miRNA miRNA 156 increasing leaf initation, decreasing apical dominance, and forming bushier plant. miRNA 164 stamens are fused together. miRNA 172 sepal and petal disappeared. miRNA 319 Leaf morphology Slide courtesy of Baohong Zhang, East Carolina Univ

21. Small interfering RNAs inhibit expression of a homologous gene

22. Biogenesis of miRNAs Bartel, 2004. Cell. PlantAnimal

23. Post-transcriptional gene regulation Two major molecular mechanisms Mechanisms of miRNA-mediated gene regulation Zhang et al. 2006. Developmental Biology Slide courtesy of Baohong Zhang, East Carolina Univ

24. Mary-Dell Chilton Undergrad and PhD University of Illinois Postdoc with Gene Nester and Milt Gorgon Univ Washington One of the first plant transformation Washington University Career at Ciba  Novartis  Syngenta

25. Pre-transcriptional gene regulation by methylation of DNA and acetylation of histones

26. Special proteins (e.g. chromomethylases) maintain methylation patterns

27. Switching a gene on and off through DNA methylation and histone modification

28. Arabidopsis MET1 Cytosine Methyltransferase Mutants Kankel et al. 2003. 163 (3):1109 Genetics Plants mutant for MET1 show late-flowering phenotypes

29. Histone acetyl transferases and chromatin remodeling allows promoters to be accessible to RNAPII

30. Histone tails are modified and can be studied easily

31. Figure 6.9

32. Some post-translational modifications Phosphorylation Biotinylation Glycosylation Acetylation Alkylation Methylation Glutamylation Glycylation Isoprenylation Lipoylation Phosphopantetheinyl ation Sulfation Selenation C-terminal amidation

33. Phosphorylation is important for intracellular signalling http://www.scq.ubc.ca/wp-content/uploads/2006/07/phosphocascades.gif

34. Protein glycosylation in the ER

35. The central dogma revisited The order of the DNA template or coding strand is 3’ to 5’ This determines the order of the mRNA strand (5’ to 3’) because DNA template is complementary to the mRNA strand.

36. Figure 6.5 Eukaryotic gene structure and transcription of DNA into mRNA TF AAAAA RNA polymerase II TF

37. Manipulating gene expression Can be done at several levels –Promoters, enhancers, transcription factors –Post-transcriptional –Translational –Methylation Biotechnology typically manipulates promoter Post-transcriptional gene silencing (RNAi) increasingly important