CHAPTER 26 RNA Metabolism

1. CHAPTER 26RNA Metabolism • Transcription: DNA-dependent synthesis of RNA • RNA processing • RNA silencing, RNA-dependent RNA and DNA polymerases Key topics:

2. Overview of RNA Function • Ribonucleic acids play three well-understood roles in living cells • Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell • Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis • Ribosomal RNAs are the constituents and catalytic appropriate amino acid • Ribonucleic acids play several less-understood functions in eukaryotic cells • Micro RNA appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences • Ribonucleic acids act as genomic material in viruses

3. Overview of RNA Metabolism • Ribonucleic acids are synthesized in cells using DNA as a template in a process called the transcription • Transcription is tightly regulated in order to control the concentration of each protein in the cell at optimal level • Being mainly single stranded, many RNA molecules can fold into compact structures with specific functions • Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors • Most eukaryotic ribonucleic acids are processed after synthesis • Elimination of introns; joining of exons • Poly-adenylation of the 3’ end • Capping the 5’ end

4. Transcription in E. coli • Substrates: Nucleoside triphosphates add to the the 3’ end of the growing RNA strand • Template: strand is DNA • Enzyme: RNA polymerase

5. Replication vs. Transcription • Both add nucleotides via an attack of the 3’ hydroxyl of the growing chain to -phosphorus of nucleoside triphosphates • RNA synthesis requires ribonucleoside triphosphates • RNA synthesis pairs A with U instead of dA with dT • Cis Site = Origins • Both require catalysis by a Mg++-dependent enzyme • RNA synthesis has lower fidelity • RNA synthesis does not require a primer for initiation • Cis Sites = Promoters • Both require a single strand of DNA as molecular template for building the new strand

6. Both DNA Strands may Encode for Proteins • Adenovirus is one of the causative agents of common cold • Adenovirus has a linear genome • Each strand encodes for a number of proteins

7. RNA Synthesis is Catalyzed by the RNA Polymerase • Mg++ on the right coordinates to the -phosphate and stabilizes the negatively charged transition state

8. Movement of RNA Polymerase Causes Local Supercoiling • Positive supercoils (overwound) ahead of the bubble • Negative supercoils (underwound) behind the bubble • Topoisomerase eliminates positive supercoils

9. Bacterial RNA Polymerase has at Least Six Subunits • Two two  subunits function in assembly and binding to UP elements • The  subunit is the main catalytic subunit • The ’ subunit is responsible for DNA-binding • The  subunit directs enzyme to the promoter • The  appears to protect the polymerase from denaturation

11. Promoter “Consensus” Sequences in DNA • Typical promoters have TATA sequence at -10 base pairs (before the transcription start site - +1) • -35 Hexamer TTGACA; Other less common sequence elements in very strong promoters

12. Alternative  Subunits Determine Promoter Specificity

13. Transcription Initiation in E. coli • In the closed complex, the DNA in the promoter region is bound to polymerase but not unwound • In the open complex, the two chains in the AT-rich promoter region region are separated • subunit leaves before elongation starts

14. Termination: RNA secondary Structure Affects Processivity • RNA polymerase “pausing” • Elongation rate reduced. • RNA/DNA/Polymerase ternary complex • Inverted-repeat sequences • Hairpin within the product • Or heteroduplex with Nontemplate DNA • RNA-DNA hybrid is disrupted • Stalling promotes dissociation of the polymerase

15. Eukaryotes Contain Several Distinct Polymerases • RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs) • RNA polymerase II is responsible for synthesis of mRNA • Very fast (500 – 1000 nucleotides / sec) • Specifically inhibited by mushroom toxin -amanitin • RNA polymerase III makes tRNAs and some small RNA products • Plants appear to have RNA polymerase IV that is responsible for the synthesis of small interfering RNAs • Mitochondria have their own RNA polymerase

16. Eukaryotic Pol II (mRNA) General Transcription Factors

18. Pol II Complex Assembly in vitro • Assembly is initiated by interaction of TATA-binding protein (TFIID) with the promoter • Helicase activity in TFIIH unwinds DNA at the promoter • Kinase activity in TFIIH phosphorylates the polymerase allowing the latter to escape the promoter

19. TATA-Binding Protein Binds in the Minor Groove (Unusual!)

20. RNA processing is Co-transcriptional

21. RNA Processing • Almost all newly synthesized RNA molecules (primary transcripts) are processed to some degree in eukaryotic cells • The 5’-end is capped w/ methylguanosine • Introns are spliced out • Poly-A tail is built at the 3’ end • Processing is catalyzed by protein-based enzymes and by RNA-based enzymes (ribozymes) • Only some prokaryotes have to splice out introns but many process their tRNA precursors

22. 5’ mRNA Cap

23. C-terminal Domain (CTD) of Polymerase II • Repeated 7-mer • Tyr-Ser-Pro-Thr-Ser-Pro-Ser • Phosphorylated • Other RNA processing enzymes loaded via P-CTD as well

24. Four Major Groups of Introns • Spliceosomal introns are spliced by splicesomes • These are most common introns • Frequent in protein-coding regions of eukaryotic genomes • Group I and Group II introns are self splicing • Interrupt mRNA, tRNA and rRNA genes • Found within nuclear, mitochondrial, and chloroplast genomes • Common in fungi, algae, and plants, also found in bacteria • Group I and Group II differ mainly by the splicing mechanism • tRNA introns are spliced by protein-based enzymes • Found in certain tRNAs in eukaryotes and archae • Primary transcript cleaved by endonuclease • Exons are joined by ATP-dependent ligase

25. Splicing of Group I Introns

26. Splicing mechanism of group II introns

27. Introns Defined by Consensus Sequences • Sequences Complementary to Small Nuclear RNAs snRNAs

28. mRNA Splicing (1) • snRNAs live in snRNPs (RibonucleoProtein Particles) • Sequential assembly of snRNPs to form the Spliceosome

29. mRNA Splicing (2) • Activation of the internal branchpoint Adenine • Attack on the 5’ splice site • Lariat Formation • Attack of the 5’ Junction on the 3’ Splice site

30. Spliceosome/CTD association • Spliceosome components also associate with CTD • Co transcriptional processing • Efficient localization to mRNA

31. PolyAdenylation • Poly A tail at 3’ end • Consensus RNA Cleavage Sequence • PAP complex loaded from CTD • Endonuclease • Poly A Polymerase • Not template directed

32. Eukaryotic mRNA processing

33. Alternative Processing generates several protein isoforms from a single gene

34. Stable RNA Synthesis • rRNA • Synthesized by Pol by • Processed I in the nucleolus by snoRNAs • tRNA • Synthesized as longer precursors • Both contain modified bases (post-transcriptional)

35. microRNAs Regulate mRNA Stability and Transcription • Complementarity to mRNA • Anti-viral and regulatory functions • RNA induced Silencing Complex (RISC) • Dicer endonuclease • RNA helicase • Polymerase, Rdp • RNA induced Transcriptional Silencing Complex (RITS) • Nuclear Complex targets chromatin

36. Tiled Genomic MicroArray – Complete Coverage

37. TranscriptomeAnalysis • mRNA small fraction of total RNA • cDNA amplification by primer extension • Fluorescent labeling • Hybridization to tiled array • Expression levels by tissue, developmental stage, disease state, • RNA silencing, Chromatin Maintenance

38. Beyond the Central Dogma DNA-Directed DNA polymerase DNA –Directed RNA polymerase RNA-directed DNA Polymerase RT, Telomerase RNA directed RNA Polymerase SiRNA RDP

39. Retroviral Infection • RNA Genome • Protein Coat • Membrane Envelope • RT Packed in the virion • ReverseTranscription Makes dsDNA • Integrase (site specific Recombinase) catalyzes Insertion

41. Retroviral Replication • Promoters in LTRs • RNA transcript encodes a poly protein • Poly-Proteins Processed • mRNA processed distinct from Genomic RNA

42. Rous Sarcoma Virus carries an OncogeneHIV encodes specialized regulators

43. Retro-elements similar in sequence, organization and function • Mobile elements replicate infrequently • within, not between genomes

44. Telomerase • RNA directed DNA polymerase • Ribonucleoprotein complex • Internal RNA template for DNA Synthesis • Inchworm model for synthesis of DNA repeats

46. Chapter 26: Summary In this chapter, we learned that: • RNA polymerase synthesizes RNA using a strand of DNA as a template and nucleoside triphosphates as substrates • The primary RNA transcript in eukaryotes requres processing before it becomes messanger RNA • The processing involves capping 5’end with methylguanosine to stabilize the RNA molecule • The processing involves splicing out introns • Some introns have an amazing ability to carry out their own splicing