Biochem - Unit II - Nucleic Acids

  1. Define a DNA polymerase reaction and its components.
    template, enzymes, dNTP, Mg++
  2. Where does DNA replication begin? Where does it terminate?
    • Ori C
    • TUS in pro
    • end of the strand in euk
    • AZTTP for retro
  3. Know about leading and lagging strand synthesis.
    • leading: continuous synthesis, fast, poly III, one primer
    • lagging: discontinuous, slow, poly III, poly I, DNA-ligase, multiple primers
  4. How many primers are required for each strand synthesis?
    1 vs 100,000
  5. Primer (DNA or RNA) with 3’OH, a minimum of __ bases hydrogen bonded to template
  6. What are Okazaki fragments? Remember some of the components required in lagging strand synthesis such as Pol I to _______ and DNA ligase to _______.
    • short segment of DNA made on lagging strand
    • remove RNA primers and add dNTP one by one
    • join the Okazaki fragments
  7. Know the polarity of DNA to be replicated.
    • copying starts w/ 3'-end
    • extend 5'->3'
  8. Know the major eukaryotic DNA polymerases.
    • I: slow, one-by-one, small, exnuclease
    • III: large, fast, complex
    • mtDNA pol
  9. Remember clamp protein (____ in eukaryotes and ____ in prokaryotes) that _________.
    • PCNA
    • β clamp
    • clamps DNA pol (mainly delta and epsilon) to template and speeds up polymerase rate
  10. What does enzyme telomerase do? It has RNA component with sequence complimentary to telomeric( DNA) sequences.
    • fix the 5' end problem
    • use RNA template to extend the original template much beyond
    • then use the extended DNA as the template to resume the duplication to guarantee the original parental info is copied.
  11. _____ that convert supercoiled DNA to relaxed form
  12. ______ that separate two parental strands of DNA
  13. Accessory proteins promoting tight binding of polymerase to DNA and thereby _______ (sliding clamps)
    increase the speed of polymerases
  14. Extension of RNA primer
    DNA polymerase III
  15. Removal of RNA primer and replacing w/ dNTP
    DNA polymerase I, one nucleotide at a time
  16. sealing of the nick for lagging strand
  17. Open the DNA double helix
    DNA helicase
  18. single-stranded DNA-binding protein
    SSB, stabilizing the separated single-strand parental DNA before their daughter copies are produced
  19. OriC
    • origin of replication (also called the replication origin) is a
    • particular sequence in a genome at which replication is initiated
  20. dnaA:
    [duplication initiation factor, binds to] Ori C
  21. dnaB:
    begins unwinding (helicase)
  22. Gyrase and Topoisomerase:
    Supercoil to relax transition
  23. Primase:
    Synthesis of primer (RNA)
  24. Termination of cyclic dsDNA synthesis
    • termination signal: TUS factor
    • Two forks reach each other at mid point of the circle and are stalled by TUS factor
    • Replication completes with generation of catenated circles, which are separated by Topo IV
  25. PROOF READING ACTIVITY OF POL I is acting as a
    3’ to 5’ exonuclease
  26. E.coli pol I structure
    • Thumb
    • Palm
    • Fingers
    • 3’ – 5’ exonuclease
  27. cell cycle
    • G1: starts with one copy
    • S phase - synthesis, duplicating
    • G2: two copies made
    • M: mitosis
  28. unique Problem with Eukaryotic DNA replication and solution
    • 5’ – RNA primer removal and fill up
    • telomerase
  29. Define RNA polymerase reaction. How does it differ from DNA pol reaction?
    • no primer
    • different polymerases
  30. What are pro and eukaryotic promoter sequences? How are these recognized?(e.g. sigma factor)
    • pro
    • tataat pribnow box
    • ttgaca
    • by sigma factor of RNA polymerase

    • euk
    • tata, gcgc, caat
    • by transcriptional factors
  31. What is the termination signal for RNA synthesis in prokaryotes?
    poly-U, hair pin, rho-factor - C-rich region
  32. How many eukaryotic RNA polymerases exist and what do they synthesize?
    • 3
    • I: rRNA
    • II: mRNA
    • III: tRNA
  33. What is the one major difference in prokaryotic and eukaryotic transcription? How is primary transcript processed in eukaryotes?
    • Eukaryotes: mRNA sites in nucleus, then it comes out­
    • Prokaryotes: process where it is made and then used and binds to ribosomes­
    • Primary transcript: eukaryotes do this, prokaryotes don’t
    • polycistronic vs monocistronic
  34. Remember capping at 5’end and poly A addition at 3’ends. Also know the processing of intron-exon sequences.
    • snRNPs
    • splicesome
  35. Which small RNAs do you recognize? Micro RNAs, Si RNAs, SnRNP, SL7 RNA
    • miRNA: stem loop precursor, suppress
    • siRNA: RNaseIII, silencing
    • 7SL RNA: transport secretory protein
    • snRNP: mRNA processing
    • oligoA: interferon
  36. Recognize rifamycin and alpha-amanitin targets.
    • RNA polymerase (prokaryotic)
    • RNA poly II (eukaryotic)
  37. Prokaryotic RNA polymerase
    • holoenzyme: a, a, b, b', w, s, s is used to locate the start point
    • core enzyme: a, a, b, b', w, elongation of RNA chain
  38. Major RNA species
    • rRNA: Makes ribosomes
    • tRNA: carry amino acids
    • rRNA and tRNA account for 95% of all RNA
    • mRNA: short, lots more of them
    • small RNAs, U1-U6: uridine rich sequences; Participate in the splicing of RNA reactions in eukaryotes
    • miRNAs: 15-20 nt very small, missed detection
  39. Eukaryotic promoter-binding transcription factors: ______.
    CTF, SP1, and TFIID
  40. posttranscriptional modification of eukaryotic mRNA
    • 5'-5' linkage of 7-methylG-cap
    • 3' polyA tail
    • splicing of the introns (snRNP needed, splicesome formed)
    • RNA polymerase I : rRNA; in Nucleoli
    • RNA polymerase II: mRNA; Nucleoplasmic
    • RNA polymerase III: tRNA, 5S rRNA; Nucleoplasmic
    • -110 -60 -25
    • CAAT GCGC TATA (box)
  43. prokaryotic mRNA can be
  44. miRNAs (Micro-RNAs):
    • typically 21 to 25 base long and is produced from stem loop structured precursor RNAs.
    • Involved in destruction of specific gene transcripts and suppression of translation of specific mRNAs.
  45. siRNA or RNAi:
    • small interfering RNAs similar to miRNAS, produced by RNase III enzyme
    • Functions in Silencing of mRNA
  46. 7SL RNA:
    • RNA required in transport (to Golgi) of secretory
    • proteins.
  47. SN-RNA:
    (small Nuclear RNAs) form RNPs required in processing of mRNA.
  48. Oligo A:
    (20-50 nucleotide long) stretch of A nucleotides linked from 2’ to 5’. Aids in interferon action.
    • Synthesized as long transcripts
    • 16 S tRNA 23S 5S
    • Cleaves at spacer sequences with specific RNase
    • CCA ends are added to the 3’-ends of all tRNAs
    • Modifications of some bases, e. g. methylation in both tRNA and rRNA
    1. Enzyme specific
    2. DNA intercalating agents
    3. Substrate analogs
    • Rifamycin for E. coli enzyme
    • a-amanitin for pol II

    • Actinomycin D
    • Quinacrine

    • Ara - C
    • Ara - A
  51. Down-regulation of the supply of initiator Met-tRNAi via ______
    eIF2 kinases
  52. Puromycin
    • causes Premature release of nascent
    • polypeptide by imitating tyrosinyl-tRNA and targeting 50S, 60S
    target 30S and Inhibits initiation
    target 30S and Inhibits binding of AA-tRNA to A-site
    Inhibits peptidyl transferase via targeting 50S
  56. Erythromycin
    Inhibit translocation via targeting 50S
  57. RICIN (castor beans)
    targets 60S and Inhibits binding of AA-tRNA to A-site
  58. Diphtheria toxin
    inactivates eEF2 for ribosome translocation
  59. Formation of aminoacytyl-tRNA
    • AA+ATP+aminoacytyl-tRNA synthetase->enzyme(aminoacytyl-AMP)+PPi->2Pi
    • enzyme(aminoacytyl-AMP)+tRNA->AMP+enzyme+aminoacytyl-tRNA
  60. Protein Modifications
    Hydroxylation: (Proline) in collagen, Endoplasmic Reticulum

    Glycosylation: O-linked as with Ser/Threo- OH, in Golgi; N-Linked as in lysine, in ER
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Biochem - Unit II - Nucleic Acids
Biochem - Unit II - Nucleic Acids - Modak