2. RNA: Structure & Synthesis By Amr S. Moustafa, M.D.; Ph.D.

3. Objectives  The differences between DNA and RNA  The structure and functions of RNAs  RNA synthesis (Transcription)  Post-transcriptional events (modifications)

4. RNA & DNA: Similarity Both RNA & DNA:  Unbranched polymers  Nucleoside monophosphate  Phosphodiester bonds

5. RNA & DNA: Differences RNA Single-Strand (mostly) Cytoplasm (mainly) AGCU (exception) Modified bases Ribose Protein Biosynthesis Post-transcriptional events DNA Double Nucleus d AGCT Deoxyribose Storage &transfer DNA Repair

6. Gene Expression - 1

7. Gene Expression - 2 From Nucleus to Cytoplasm DNA(Gene) (1) mRNA (2) Protein Information Pathway

8. RNA: Types Major types: Ribosomal RNA (rRNA) 80% Transfer RNA (tRNA) 15% Messenger RNA (mRNA) 5% Others: Small nuclear RNA (snRNA)

9. Nucleoprotein complexes of ribosomes Svedberg Unit: The rRNA Related to Molecular weight & Shape

10. The tRNA  Smallest RNA 4S (74 – 95)  At least 20 species  Unusual bases  Secondary structure Intra-chain base pairing  Adaptor molecule Carries its sp. a.a. to site of protein biosynthesis

11. The mRNA  Size:Heterogeneous (500 – 6000)  Primary (precursor): hnRNA  Post-transcriptional Processing of Euokaryotic mRNA  Carries genetic information from nucleus to cytoplasm (Template of protein synthesis)

12. RNA Synthesis (Transcription)  Transcription: the copying process which uses one of the two DNA strands (template strand) to form RNA  Other DNA strand: coding strand Similar to RNA strand (with exception)  Transcription is highly selective: Some DNA regions form many, other form few or no transcripts

13. Transcription - 2  The selectivity is due to signals in the DNA nucleotide sequence  The signals convey a message to RNA polymerase to define: Where polymerase should start? How often? (frequency) Where to stop? (termination)

14. Transcription – 3 Prokaryotic RNA polymerase

15. Transcription – 4 Prokaryotic RNA polymerase  One species for all RNAs (Except primase for RNA primer) Elongation: 5’– 3’ polymerizing activity Initiation: Recognition of promoter sequence Termination: ρ(rho)-independent  No exonuclease or proofread activity  Transcription:

16. Transcription – 5 Complementary and antiparallel DNA and RNA

17. Transcription – 6 Prokaryotic Promoter  Promoter is DNA sequence recognized by (σ) Sigma factor of RNA polymerase holoenzyme where transcription should start  Consensus, highly conserved sequences -10 Pribnow box (TATAAT) -35 Sequence (TTGACA) (as read 5’ –3’ on nontemplate ‘coding’ strand)  Mutant promoter affects transcription of its gene

18. Transcription – 7 Prokaryotic Promoter Consensus, highly conserved sequences

19. Transcription – 8 Transcription Unit  Transcription Unit: DNA region from promoter to termination  The product: primary transcript

20. Transcription – 9 Initiation Binding of RNA polymerase holoenzyme to promoter Local unwinding Supercoils (Topoisomerases I & II) Denovo synthesis: No primer required Substrate: Ribonucleoside triphosphate Usually begins with a purine No proofreading Once promoter is recognized, Sigma (σ) factor is released

21. Transcription – 10 Elongation Direction: 5’– 3’, antiparallel Complementarity: as DNA, but for A there is U release of pyrophosphate Relaxation of supercoils: Topoisomerases I and II

22. 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

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

24. 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)

25. Transcription – 14 Rifampin inhibits RNA polymerase and transcription