Introduction to Transcriptional Machinery

1. Introduction to Transcriptional Machinery

2. "DNA makes RNA, RNA makes protein, and proteins make us." Francis Crick

3. Central Dogma of Molecular Biology

4. RNA Polymerase of E. Coli • Transcribes all mRNA, rRNAs and tRNAs • 7,000 molecules per cell • 5,000 molecules are synthesizing RNA at any given time • M.W. of the holoenzyme is ~465 Kd

5. RNA Polymerase of E. Coli

6.  Factor Controls Specificity

7. Holoenzyme & Core Enzyme • Holoenzyme binds promoters with half lives of hours - 1,000 time higher than core enzyme. • Holoenzyme has a drastically reduced ability to recognize “loose binding sites” - half life of <1sec – 104 time lower than core enzyme.

8. Transcription Initiation

9. Promoter Elements in E. Coli • -35: recognition domain • -10: unwinding domain • Seperating distances • UP element • Start Point: purine in 90% of the genes 16-19

10. First Level of Regulation • ~100 fold variationin the binding rate of RNA Pol to different promoters in vitro. • Binding rates correlate with the frequencies of transcription in vivo. T80A95T45A60A50T96

11. E. Coli has several  Factors

12.  Factors Recognize Promoters by Consensus Sequences

13. Termination

14. What was known in the 1960’s • Jacob and Monod 1961 – genetic control mechanisms in prokaryotes • Anticipation for Eukarotes… • Eukaryotes – genomic complexity – reiterated DNA sequences • Lack of genetic approach

15. February 1969, Strait of Juan de Fuca

16. “Eureka!” Taken from: The eukaryotic tarnscriptional machinery, Robert G Roeder

17. 3 RNA Polymerases • Pol I localized within nucleoli – the sites of rRNA gene transcription • Pol II and Pol III restricted to the nucleoplasm

18. 3 RNA Polymerases • Roberto Weinmann - 1974 • Differential sensitivities to the mushroom toxin  - amanitin • Pol I – rRNA synthesis • Pol II – adenovirus pre-mRNA • Pol III – cellular 5S and tRNA

19. RNA Polymerases of Eukaryotes • Pol I - transcribes pre-ribosomal RNA (18S, 5.8S, 28S) • Pol II - mRNAs • Pol III - tRNAs, 5S RNAs and some specialized small RNAs.

20. RNA Polymerase II • 2002 – RNA Pol II structure • 2003 – transcription complex structure (RNA Pol II + TFIIS) • , ’, I, II,  - conserved in yeast and bacteria – evolutionary conserved mechanism of transcription

21. Significant homology between eukaryotic and bacterial RNA polymerases in their structure

22. Transcription Mechanism • RNA Pol II can catalyze RNA synthesis but cannot initiate. • Assembly • Initiation • Elongation • Termination

23. Transcription Mechanism

24. TBP • Only GTF that creates sequence specific contact with DNA • Unusual Binding in minor groove • Causes DNA bending

25. TBP • 80% conserved between yeast and man • Large outer surface binds proteins • Deformation of DNA structure, but no strand separation

26. The transcriptional machinery • Initiation begins with the formation of the first phosphodiester bond and phosphorylation of Ser5 on the CTD by TFIIH. • mRNA passes through a positively charged exit channel, and once the RNA is approximately 18n long it becomes accessible to the RNA processing machinery. • Consistent with the coupling of transcript capping to early transcription events

27. Pre-mRNA Processing • Addition of 5’ cap • Splicing – removal of intron sequences • Generation of 3’ poly-A tail. • 3’ cleavage • RNA serveillance by the exosome • Packaging of the mRNA for export Occurs (most efficiently) co-transcriptionally

28. Transcription Regulating Elements • GTFs - required at any Pol II promoter • Enhancers – sequences, increase transcription • Transactivators - bind enhancers • Co-activators - act indirectly, not by binding to DNA, communication between transactivators and RNA PolII + GTS • Mediator - 20 proteins, Interacts with CTD

29. Major Differences between Pro & Eu • Prokaryotes RNA Pol has access to promoters and initiates transcription even in the absence of activators and repressors. • Eukaryotes - promoters are generally inactive in vivo • Transcription in eukaryotes is seperated in both space and time from translation

30. The CTD is Phosphorylated at Initiation

31. CTD • Highly conserved tandemly repeated heptapeptide motif (YSPTSPS) • Platform for ordered assembly of the different families of pre-mRNA processing machinery • Undergoes phosphorylation and dephosphorylation during the transcription cycle

32. CTD • P-TEFb contains CDK9 and cyclin T • It couples RNA processing to transcription by phosphorylating Ser2 of CTD • RNA Pol II is recycled through dephosphorylation of Ser2 by the phosphatase activity of Fcp1

33. CTD Phosphorylation During Transcription

34. Splicing (& Alternative Splicing)

35. Expansive role of Transcription • RNA surveillance – Exosome associates with Spt6 EF • Coupling of transcription to mRNA export • 19S particle of the Proteosome recruited to active promoters – important for efficient RNA Pol II elongation

36. Translation and Post-Translation • Bacteria – translation occurs as the nascent transcript emerges from the RNA polymerase • It is assumed that in eukaryotes transcription and translation are spatially separated events • Protein synthesis – solely a cytoplasmic event? (1977 – Gozes et al, 2001 lborra et al)

37. Traditional View of Gene Expression

38. Contemporary View of Gene Expression

39. The Sister Chromatids of a Mitotic Pair

40. Chromatin Packing 2 nm 105mm Double helix 11nm “Beads-on-a-string” ~x7 30 nm fiber of Packed nucleosomes ~x100 30 nm Chromosomal loops Attached to nuclear scaffold 300 nm Condensed section of metaphase chromosome 700 nm ~x104 Entire metaphase chromosome 1400 nm 5-10 mm

41. GC Pairs Are Preferred DNA Histone Core AT Pairs Are Preferred Chromatin Structure • DNA accessibility – a major challenge in a chromatin environment • Nucleosomes – building blocks of chromatin

42. Structure of the Nucleosome • 146 bp are wrapped around the histone core • 1.75 times • ~0-80 bp in the linker sequences between nucleosomes • Human genome (~6x109 bp) contains ~3x107 • nucleosomes • The histone core (octamer) consists of two copies of: • Histones H2A, H2B, H3 and H4 • Histone H1 binds in the spacing linker sequence

43. The Nucleosome DNA H2B Histone Core H2A H4 H3 H3 H2B H2A

46. Histones • Highly conserved throughout eukaryotic evolution • Mutations in histones encoding genes are often lethal • Highly abundant (~60 million copies/cell) • Additional non-histone proteins play a role in the chromatin structure and function

47. Types and Properties of Histones

48. Interaction of DNA with Positively Charged Residues in the Nucleosome Core DNA Red: The positively charged lysines & arginines The DNA is wrapped along these residues

49. H1 Histone • In the presence of H1, 166 bp • are protected from nucleolytic cleavage -> • full two tight loops (83 x 2 bp). • When histone H1 is extracted, the resulting structure is the 11 nm “beads-on-a-string”

50. View Along the Axis of One Turn of the 30nm Fiber DNA Histone H1 Histone Core