Transcription and Splicing machinery

1. DNA  primary mRNA  mature mRNA Transcription + Processing Transcription and Splicing machinery

2. Prokaryotic and Eukaryotic RNA Polymerases are similar in shape Sigma (σ) subunit missing -> Different number of subunits

3. Recognizes the promoter site (-10 box + -35 box)

4. RNA polymerase mechanism -> Similar to DNA polymerase -> 3’-hydroxyl group of RNA chain attacks the a-phosphoryl group of the incoming NTP -> Transition state stabilized by Mg2+

5. Transcription AFM image of short DNA fragment with RNA polymerase molecule bound to transcription recognition site. 238nm scan size. Courtesy of Bustamante Lab, Chemistry Department, University of Oregon, Eugene OR

8. Prokaryotic promoter sites -35 -10 +1 5’-----TTGACA--------------TATAAT---------start site----3’ σ subunit

9. Prokaryotic promoter sites σ subunit interacts with -10 box and -35 box

10. Alternative E. coli promoters Stanard Promoter -> σ70 Heat shock promoter -> σ32 N-starvation promoter -> σ54

11. Footprinting

12. DNA unwinding prior to Initiation of Transcription -> Transition from closed to open complex -> Unwinding done by RNA polymerase 1 RNA polymerase molecule -> 17bp segment -> 1.6 turns on B-DNA

13. Negative supercoiled DNA favors the transcription -> neg. supercoiling facilitates unwinding -> introduction of neg. supercoiling -> increases rate of transcription -> Exception -> promoter of TopoII -> neg. Supercoiling -> decreases rate of transcription

14. Transcription bubble First Nucleotide is pppG or pppA -> Transcription start

15. RNA-DNA hybrid separation RNA polymerase forces the separation of the RNA-DNA hybrid

16. Transcription Termination Rho independent termination Termination by Rho protein -> RNA polymerase pauses after production of hairpin -> RNA-DNA hybrid of hairpin is unstable => RNA falls off Rho interacts with RNA polymerase -> breaks the RNA-DNA hybrid helix -> functions as a helicase

17. Primary transcript of rRNA is modified Modification: 1. Cleavage of primary transcript by Ribonuclease III 2. Modification of bases (Prokaryotes: methylation) and ribose (Eukaryotes: methylation)

18. tRNA transcript is also modified Modification: 1. Cleavage of primary transcript by Ribonuclease III 2. Addition of nucleotides at 3’ end (CCA) 3. Unusual bases

19. tRNA transcript processing Modification: 1. Cleavage of primary transcript by Ribonuclease III 2. Addition of nucleotides at 3’ end (CCA) 3. Unusual bases

20. Antibiotic Inhibitors of Transcription Rifampicin: - derivate of rifamycin (Streptomyces) - inhibits initiation of RNA synthesis (binds to RNA polymerase -> in pocket where RNA-DNA hybrid is formed) Actinomycin D: - polypeptide-containing (Streptomyces) - binds tightly (intercalates) to ds-DNA (cannot be template for RNA synthesis) - its ability to inhibit growth of rapid dividing cells makes it a effective agent in cancer treatment

21. Transcription and Translation in Prokaryotes and Eukaryotes

22. α-Amanitin: produced by mushroom (Amanita phalloides) -> cyclic peptide of 8 amino acids -> binds tightly to RNA polymerase II -> blocks elongation of RNA synthesis -> deadly doses (LD50 is 0.1 mg/kg)

23. Different Eukaryotic RNA Polymerase promoters Inr -> Initiator element (found at transcription start) DPE -> downstream core promoter element

24. Eukaryotic promoter elements (RNA polymerase II promoter) -> -40 and -150 Normally between -30 and -100 Often paired with Inr -> -3 and -5 CAAT boxes and GC boxes can even be on noncoding strand active DPE -> +28 and +32

25. Eukaryotic Transcription Initiation TappingMode AFM image of an individual human transcription factor 2: DNA complex. Clearly resolved are the protein:protein interactions of two transcription factor proteins which facilitate the looping of the DNA, allowing two distal DNA sites to be combined. AFM provided the investigators' improved resolution of the looped DNA complexes compared to electron microscopy of rotary shadowed samples. 252 nm scan. Image courtesy of Bustamante Lab, Institute of Molecular Biology, University of Oregon, Eugene.

26. Eukaryotic Transcription Initiation Basal transcription apparatus (-> carboxylterminal domain) TATA-box binding protein (TBP is a component of TFIID) recognizes the TATA box and forms complex with DNA CTD plays a role in transcrition regulation -> binds to mediator Phosphorylation of CTD by TFIIH -> elongation of transcription

27. Eukaryotic Transcription Initiation Complex

28. Regulation of Transcription

29. Packaging of Eukaryotic chromosomal DNA

30. Transcription Initiation

31. Gene “Off” Gene “On”

33. Eukaryotic transcription products (from RNA polymerase II) are processed triphosphate 7-methylguanylate end Polyadenylation of 3’ end Capping 5’ end

34. RNA editing

35. Splicing Anemia: defect synthesis of hemoglobin Mutations affecting splice sites cause around 15% of all genetic diseases Creates a new splice site

37. Small nuclear RNAs in spliceosomes catalyse the splicing

38. Spliceosome assembly The catalytic center of the spliceosome

39. Alternative splicing

40. Self-splicing A rRNA precursor of Tetrahymena (protozoan) splices itself in the presence of guanosine (G) as co-factor The L19 RNA is a intron that is catalytical active This TappingMode scan of the protozoan, Tetrahymena, shows its cilia-covered body and mouth structures. The sample was dried onto a glass slide and scanned; no other preparation was required. 50 micron scan courtesy C. Mosher and E. Henderson, BioForce Laboratory and Iowa State University.

41. Self-splicing mechanism

43. Ribosomal Factory Protein mRNA Translation

44. Translation: mRNA -> Protein

45. Peptide bond formation in Ribosomes

48. Linkage of Amino Acids to tRNA 2nd step 1st step Linkages either 2’ or 3’ 1st step: activation of AA by adenylation (Aminoacyl-AMP) 2nd step: linkage of AA to tRNA

49. Aminoacyl-tRNA synthetases couple Amino acids to tRNA Synthetases are highly specific for the amino acid (error rate 1 in 105)