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

Transcription

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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 Information Pathway From Nucleus to Cytoplasm DNA(Gene) (1) mrna (2) Protein

8 Major types: RNA: Types Ribosomal RNA (rrna) 80% Transfer RNA (trna) 15% Others: Messenger RNA (mrna) 5% Small nuclear RNA (snrna)

9 The rrna Nucleoprotein complexes of ribosomes Svedberg Unit: 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 Size:Heterogeneous ( ) Primary (precursor): hnrna Post-transcriptional Processing of Euokaryotic mrna The 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) No exonuclease or proofread activity Transcription: Initiation: Recognition of promoter sequence Elongation: 5 3 polymerizing activity Termination: ρ(rho)-independent

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