2. RNA Synthesis (Transcription) By Amr S. Moustafa, M.D.; Ph.D.

3. Prokaryotic Transcription (Summary) RNA Polymerase: DNA-dependent RNA polymerase Initiation: Promoter Region Transcription Unit Elongation: 5’-3’ direction Termination: The rho (ρ)-dependent termination The rho (ρ)-independent termination

4. Transcription – 11 Termination A-The rho ( ρ) -dependent termination:  The ρ -factor binds to C-rich near 3’-end of RNA  It has ATP-dependent DNA-RNA helicase activity Hydrolyzes ATP (Energy-dependent) Unwind 3’-end of the transcript from template  Movement of (ρ) protein along RNA/DNA hybrid 5’-3’  Displacement of DNA template at termination site, releasing of RNA transcript

5. Transcription – 12 Termination (Cont’d) B-The rho-independent termination:

6. Transcription – 13 Inhibitors (Antibiotics) Rifampin (anti-tuberculous): binds to the β-subunit interferes with formation of first phosphodiester bond inhibit RNA synthesis and bacterial growth Dactinomycin (Actinomycin D): binds to minor groove of DNA template interferes with movement of RNA polymerase inhibits prokaryotic transcription (antibiotic) inhibits euokaryotic replication (anticancer)

7. Transcription – 14 Rifampin inhibits RNA polymerase and transcription

8. Eukryotic Transcription dsDNA: Loose conformation Dissociate temporarily from nucleosome core RNA polymerases: Several classes Both nuclear and mitochondrial Transcription factors: Proteins that bind to specific DNA sequences (within or outside promoter)

9. Chromatin structure and gene expression Euchromatin: Relatively relaxed chromatin Contains actively transceibed genes Heterochromatin: Highly condensed Inactive segment of DNA Chromatin remodeling: Interconversion of active and inactive forms

10. Chromatin structure: Regulation DNA methylation: commonly 5 methylcytosine More DNA methylation; Inactive genes One of female X-chromosome Highly methylated, heterochromatin Histone acetylation: Acetylated histones, looser chromatin active genes for transcription

11. The eRNA polymerases A. Nuclear: 3 Classes, I, II & III Large enzymes, multiple subunits B. Mitochondrial: Single species resembles bacterial RNA polymerase

12. The eRNA polymerases: Nuclear PolymeraseGenesSite I Large rRNA 28, 18 & 5.8 S Nucleolus II mRNA snRNA Viral RNA Nucleoplasm III tRNA Small rRNA (5S) snRNA Nucleoplasm

13. The eRNA polymerase II Promoter: - 25 bp: TATA or Hogness box - 70 bp: CAAT box In addition, GC box Cis-acting genetic elements (DNA sequences): on the same molecule to be transcribed binding sites for general transcription factors Trans-acting elements (proteins): Transcription factors encoded by genes on different chromosomes either stimulate or inhibit transcription

14. The eRNA polymerase II: Promoter region

15. Prokaryotic Promoter Consensus, highly conserved sequences

16. Transcription Factors General: Binds to promoter Activators: Binds to Enhancer

17. Enhancers and Silencers Cis-acting DNA sequences Enhancers: stimulate; Silencers: inhibit Upstream or downstream Close or thousands bp from promoter DNA response elements: activators-binding sites Activators can bound to either strand of DNA Bending or looping of enhancer DNA allow activators (specific TF) to interact with: General TF and RNA polymerase II

18. Enhancers

19. The eRNA polymerase II: α-amantin inhibitor Source: Amanita phalloides (death cap or destroying angel) α-amantin: forms a tight complex with the polymerase, inhibiting mRNA synthesis

20. The Lac Operon Operon: Coordinately expressed genes Structural and their regulatory genes Lac operon: (For E. coli catabolism of lactose) Structural genes: code for 3 enzymes: Lac Z gene: β-galactosidase Lac Y: permease Lac A: thiogalactoside transacetylase Regulatory Sequences: Promoter (P) region Operator (O) site CAP-binding site LacI gene: repressor protein

21. The Lac Operon: Off

22. The Lac Operon: On

23. The Lac Operon: Off