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DNA/RNA Metabolism

DNA/RNA Metabolism. Blackburn & Gait, Ch. 5 Transcription • understand components of transcription bubble • know the importance of promoter sequences in proks/euks • know how nucleic acid structure affects transcription termination (both Rho-dep and Rho-indep)

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DNA/RNA Metabolism

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  1. DNA/RNA Metabolism Blackburn & Gait, Ch. 5 Transcription • understand components of transcription bubble • know the importance of promoter sequences in proks/euks • know how nucleic acid structure affects transcription termination (both Rho-dep and Rho-indep) • know an example of activator and repressor of transcription • know what transcriptional enhancer elements also called UAS (upstream activator sequence) do and their mode of action • understand attenuation RNA Processing • understand 5’-capping reaction and reason for cap • understand 3’-endonuclease cleavage and polyadenylation and reason for A-tail • know steps in RNA splicing and alternative splicing

  2. DNA metabolism Replication DNA replication - process of copying genetic information DNA acts as a template for replication and transmission of genetic info One strand is the complement of the other

  3. DNA metabolism Stages of Replication Elongation

  4. DNA metabolism Stages of Replication Elongation

  5. RNA Metabolism Transcription - uses DNA-dependent RNA polymerase Polymerization is “asymmetric” - only one strand of DNA used as template, new RNA chain is identical in sequence to the nontemplate strand

  6. RNA Metabolism Transcription RNA synthesis initiated at promoters (specific DNA sequence) Typical E.Coli promoters: Pribnow box EUKARYOTES: -75 CAAT box -25 TATA box (TATAAA)

  7. RNA Metabolism Transcription - termination Termination sites have a palindromic sequence just prior to termination point and RNA transcribed has short inverted repeats (GC-rich regions) which form hairpin GC-rich regions followed by 4-10 A:T bp Stem-loop structure in RNA being synthesized induces pausing of RNAP Weak U:A bp cause a conformational change in RNAP and RNA breaks free of DNA Rho-independent termination

  8. RNA Metabolism Transcription - termination Rho-independent termination

  9. RNA Metabolism Transcription - termination Rho-dependent termination Also get hairpin formed but not G-C rich and no uridines after it Special protein Rho is needed for termination Rho may bind 5’-end of nascent RNA and travel along behind RNAP Rho may then interact with b-subunit of RNAP and stop transcription RHO may be a helicase and unwind the RNA from the RNA-DNA hybrid

  10. RNA Metabolism Transcription - termination Rho-dependent termination Also get hairpin formed but not G-C rich and no uridines after it Special protein Rho is needed for termination Rho may bind 5’-end of nascent RNA and travel along behind RNAP Rho may then interact with b-subunit of RNAP and stop transcription RHO may be a helicase and unwind the RNA from the RNA-DNA hybrid

  11. RNA Metabolism Transcription - frequency of transcription regulated by inhibitors (repressors) and activators of RNAP Repressor = trp repressor Activator = CAP (catabolite activator protein) Trp dimer HTH motif Trp DNA DNA Interacts with RNAP CAP dimer (HTH motif) cAMP

  12. RNA Metabolism Transcription Transcriptional enhancer elements also called UAS (upstream activator sequence) If a UAS is deleted it abolishes promoter activity

  13. RNA Metabolism Transcription Transcriptional enhancer elements also called UAS (upstream activator sequence) Enhancer function independent of orientation or distance from affected gene

  14. RNA Metabolism Transcription Transcriptional enhancer elements also called UAS (upstream activator sequence) Models for action of enhancers Enhancer box is entry site, pro binds and then migrates along DNA in order to interact with RNAP **Loop out DNA

  15. RNA Metabolism Transcription Transcriptional enhancer elements

  16. RNA Metabolism Transcription Transcriptional enhancer elements

  17. RNA Metabolism Transcription Transcriptional enhancer elements

  18. Attenuation (prokaryotes) • Attenuation is means of controlling transcription of a particular mRNA through the formation of translation-dependent alternative RNA structures • For example, expression of the operon that encodes proteins required for tryptophan biosynthesis is modulated based on supply and demand for tryptophan • A short open reading frame that encodes tryptophan, and a downstream RNA region (the attenuator) are key element of control

  19. Attenuation (prokaryotes) • Co-transcriptional translation determines what structure the attenuator RNA will adopt • Abundant supply of tryptophan and movement of the ribosome promotes a structure that terminates transcription • Stalling of ribosome due to lack of tryptophan promotes a structure that prevents termination

  20. RNA processing Proks vs. Euks In proks, transcription & translation coupled In euks, processes are temporally& spatially separated so more control

  21. RNA processing mRNA processing: 5’-capping 3’-endonuclease cleavage and polyadenylation RNA splicing

  22. RNA processing mRNA processing: 5’-capping

  23. RNA processing mRNA processing: 3’-endonuclease cleavage & polyadenylation

  24. RNA processing mRNA processing: RNA splicing & editing

  25. RNA processing mRNA processing: RNA splicing & editing

  26. RNA processing Alternative splicing

  27. RNA processing Alternative splicing

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