Plant Gene Expression

1. Genome Genes Expression Plant Gene Expression Same genome in somatic cells Different structures and functions Differential gene expression Spatial: organs Temporal: time

2. Differential Gene Expression Same proteins for Common processes Some abundant proteins in certain cell types Some minor proteins in certain cells at certain times 10,000 - 20,000 proteins in cells 15-20% as tissue-specific proteins

3. Control of Gene Expression Regulation at steps: Transcription: which gene, when, how often Post transcription: RNA processing and transport Translation: which transcript and for how long Post translation: activity and stability of proteins

4. * * * * ** * Control of Gene Expression

5. Transcriptional Control I Gene control regions & Regulatory protein components of genetic switch Gene control regions 5’ Promoter and 3’ Terminator 5’ Regulatory sequence (some inside or at 3’)

6. Proximal promoter 5’ promoter or proximal promoter Common sequence: TATA / CAAT box Binding site for transcription machinery RNA Pol and general transcription factors

7. Distal promoter Regulatory sequences or distal promoter or cis element or responsive element Specific and consensus for gene expression Binding site for gene regulatory proteins or trans acting element or trans element or specific transcription factor

8. Transcriptional Control Genetic switch: turning genes on / off

9. Transcription Initiation

10. Transcription Initiation

11. Gene activating protein DNA binding domain Gene activation domain Specific binding at major groove of DNA Control rate of transcription initiation except for general transcription factor TFIID: TATA binding protein that binds minor groove

12. Major-Minor groove

13. DNA-Protein interaction Strong and Specific binding Compatible structure of DNA and Protein 20-30 contacts per match Hydrogen bond Ionic bond Hydrophobic interaction

14. Gene Activating Protein DNA binding motif Helix Turn Helix Zn finger β sheet Leucine zipper Helix Loop Helix

15. Helix Turn Helix

16. Helix Turn Helix

17. trp repressor dimer Helix Turn Helix

18. Zn Finger

19. b sheet

20. Leucine Zipper

21. Leucine zipper

22. Helix Loop Helix

23. Transcriptional control II Chromatin structure DNA packaging Heterochromatin: condensed Euchromatin: relaxed

24. Chromatin structure Most organisms have both types Except for yeast: euchromatin some algae and maize B chromosome: heterochromatin

25. Chromatin structure

26. Chromatin structure Heterochromatin Genetically inactive Inaccessible for transcription machinery Euchromatin Loose nucleosome / movable histones Available for protein binding Genetically active

27. Chromatin structure

28. Chromatin structure

29. Transcriptional control III DNA methylation Hypermethylation: Gene inactivation Methyl group on Base Methylase

30. DNA methylation Eukaryote: more often in CG dinucleotide Vertebrate: only in CG

31. Methyl group on position #5 of Cytosine ring 5meC Cytosine Methylation

32. Maintenance of methylation pattern

33. Methylation-induced Mutation Deamination of Cytosine / 5meCytosine Cytosine deamination (U) Repair 5meCytosine deamination (T) Mutation

34. Methylation-induced Mutation

35. Methylation Plants: ~3-4 % of genome = CG Animals: ~0.5-1 % of genome = CG Methylation found in 70-80% of CG

36. Wheat Germ DNA Highly methylated on CG or CNG 82 % of CG 19 % of CA / CT 7 % of CC 80+ % of CAG / CTG 4- % of CAT

37. Post-transcriptional Control I Attenuation Bacterial regulation of polycistronic RNA Complete/Incomplete RNA production mRNA inhibit RNA polymerase incomplete transcription mRNA interact with Regulatory protein complete transcription

38. Post-transcriptional Control II Alternative splicing 1 primary transcript / many mature transcript 1 gene / diversed mRNA / many proteins May function in different organs May function in different developmental stages May have opposite functions

39. Alternative Splicing Exon skipping / Optional exon Intron retention / Optional intron Mutually exclusion exon Alternative 5’ / 3’ splice site Alternative selection of promoter / PolyA site

40. Alternative Splicing

41. Alternative Splicing

42. Alternative Splicing

43. Post-transcriptional Control III Varied C terminus Polyadenylation at different sites Same proteins of Different lengths eg. Protein with/without hydrophobic C terminus IV RNA transport Half of primary transcripts: destroyed Parts of transcripts to be processed Most mature RNA: out to cytoplasm

44. Post-transcriptional Control V RNA editing (modification) Addition or Deletion of U Deamination of C to U (plant mt) VI Trans-Splicing Exons from 2 independent transcripts

45. Post-transcriptional Gene Silencing PTGS: transcription without translation Found in plants and animals Also in protozoa, insects and nematodes Caused by transgene, virus or homologous dsRNA

46. Transgene-induced PTGS Cosuppression Silencing of Endogenous gene Triggered by Transgene Silencing occurs at post-transcriptional level Homologous transcripts made & exported Rapid degradation in cytoplasm

47. Transgene-induced PTGS Petunia Transformed with pigment-producing gene Expected deep purple color Appeared variegated to white Transgene-induced gene silencing also at transcription gene-specific methylation

48. P T P T Viral-induced PTGS Introduction of certain viruses to host plants dsRNA as trans-acting factor responsible for PTGS Antisense RNA technology for gene silencing

49. application RNA interference dsRNA to knock out gene expression so called RNA interference or RNAi by initiating Small Interfering RNA (siRNA) to induce silencing of endogenous transcript

50. RNAi Much more efficient than using either strand as in cosuppression or antisense technology Good for Gene Knockout studies easily and quickly create Loss-of-Function phenotypes Tool for Functional Genomics Study of Expressed Regions of Genome