Bio 99 Final Lec 10

  1. central dogma
    • DNA undergoes replication
    • DNA ----> RNA through transcription
    • RNA ----> Protein through translation
  2. transcription
    the process whereby genetic information in the DNA is used to create an RNA strand of complementary sequence
  3. genome
    the sum of all genetic information contained in the DNA of an organism
  4. transcriptome
    the sum of all RNA transcripts in a cell in a given moment
  5. every cell in your body had the same _____ but not every cell has the same ________
    genome; transcriptome
  6. RNA
    Ribonucleic Acid
  7. Answer Yes or No
    1.) RNA used Uracil as one of it's bases, DNA used Thymine?
    2.) RNA has a hydroxyl group on its 2' carbon, DNA does not
    3.) RNA has a hydroxyl group on its 3' carbon, DNA does not
    4.) DNA has a phosphate group on its 5' carbon, RNA does not
    • 1.) yes
    • 2.) yes
    • 3.) no
    • 4.) no
  8. RNA versus DNA structures
    • DNA can form double helix which is very regular and very stable
    • RNA is single stranded
    • RNA can also form complex secondary and tertiary structures, giving it functional capabilities
  9. RNA transcripts can serve as carriers of information from ____ to _____
    DNA; Protein
  10. mRNA
    • messenger RNA
    • contains amino acid coding information proteins
    • only 3-5% of total RNA
  11. tRNA
    • transfer RNA 
    • helps in protein synthesis
    • 15-20% of total RNA
  12. rRNA
    • ribosomal RNA 
    • helps to catalyze protein synthesis reaction
    • ~80% of total RNA
  13. other non-coding RNAs
    • smaller nuclear RNAs (snRNA)
    • long non-coding RNAs (IncRNA)
    • Micro RNAs (miRNA)
  14. Summary of Transcription (4 steps described)
    • 1.) RNA polymerase binds to double stranded DNA
    • 2.) DNA opens up to expose single strands
    • 3.) RNA polymerase read template strands and adds ribonucleotides (NTPs) to growing RNA chain
    • 4.) DNA reforms double strand and exits, RNA chain exits
  15. RNA transcription used the DNA _____ _______ as a template for transcription
    template strand
  16. Both the RNA transcript and the coding strand are the _____ ______ of the template DNA strand
    reverse complement
  17. The ____ ______ sequence is identical to the RNA sequence
    coding strand
  18. Structure of bacterial RNA polymerase core
    • 5 subunits: β, β', ω, two copies of α
    • Image Upload 1
  19. Bacteria have __ RNA polymerase while eukaryotes have ___ RNA polymerase
  20. RNA Pol ___ is most similar to bacteria
  21. Describe the transcription bubble
    • DNA duplex is unwound into single strands for a moving “bubble” of about 17 base pairs
    • Inside this bubble, the polymerase reaction occurs and the growing RNA chain is paired with the template strand to form a DNA/RNA hybrid (about 8 bp long)
    • As RNA polymerase moves through the DNA, it rotates the DNA, creating supercoils both in front and behind it
    • Supercoiling must be relieved by topoisomerases
  22. sigma factor
    • bacterial protein that links RNA polymerase to the transcriptional start site at the front of a gene
    • bacterial equivalent of transcription factors
    • sigma factors control which genes get transcribed and when
  23. common sigma factors and their functions
    • σ70- housekeeping genes, most genes
    • σ38- starvation genes
    • σ32-heat shock
    • σ54- nitrogen uptake and metabolism
  24. RNA Holoenzyme
    RNA polymerase "core" + Sigma Factor
  25. How does Sigma factors know where to find on DNA?
  26. Promoters
    • Region of DNA at the start of a gene, whose sequence determines where transcription begins and when 
    • Region where RNA polymerase binds (around 100bp, -70 to +30)
  27. Consensus Sequence
    • region in the promoter where sigma factor binds
    • -35 and -10 regions both contain consensus sequences
  28. Upstream Promoter (UP) element
    • AT-rich sequence, recognized by a subunit of RNA polymerase
    • Increase binding and transcription
  29. How do we know where sigma factors bind?
    footprinting assay
  30. footprinting assay
    bind proteins (Sigma Factors) to DNA the chop up the DNA, and see which regions are protected
  31. Footprinting Assay Steps 0-3
    • 0.) mix osmY DNA fragments radiolabeled on one end with sigma factors (or nothing)
    • 1.) Add Dnase I to cleave DNA 
    • -light treatment ensures only 1 cut per fragment
    • -randomly cleave DNA into different sized fragments
    • 2.) Remove bound protein and denature DNA 
    • -leaves naked, radiolabeled single DNA strand
    • 3.)  Run on a gel and visualize by exposure to x-ray film
    • (regions of DNA bound by sigma factor/RNA pol will not be cut)
  32. Bacterial Transcription
    • Initiation
    • Elongation
    • Termination
  33. Transcription Initiation, Part 1
    • RNA polymerase holoenzyme (core + sigma factor) binds to DNA at promoter consensus sequence. This complex is initially closed.
    • A structural change causes the DNA to melt (separate into two strands), and enter the polymerase active site, creating an transcription-competent open complex.
    • Sigma factor is blocking the exit channel for the RNA transcript
    • This process is spontaneous, reversible, and requires no energy
  34. Mechanism of the Polymerase Reaction
    • The 3’-hydroxyl group makes a nucleophilic attack on the α phosphate of the incoming rNTP, with the concomitant release of pyrophosphate
    • Image Upload 2
  35. Transcription Initiation, Part 2
    • RNA polymerase does not require a primer.
    • NTPs are added quickly, but the structure is unstable and will spontaneously stop and leave the channel. This is called abortive initiation.
    • Abortive initiation occurs frequently. If so, the process begins again until about 10 bases are added, stabilizing the transcript.
    • Sigma factor is initially blocking the exit channel for RNA. The growing transcript will dislodge sigma factor from the exit channel, causing sigma factor to release from the polymerase. Once released, the complex exits the promoter and initiation is complete. This is promoter clearance.
  36. What is the key difference between the closed and open transcription initiation complexes?

    C. )
  37. What does the nucleophilic attacking?

    A. )
  38. What is the main factor in determining whether transcription initiation completes?

    D. )
  39. Transcription Elongation:
    3 Channels in the Polymerase
    • -DNA entry channel
    • -NTP entry channel
    • RNA exit channel
    • -(no DNA exit channel)
  40. A pin region of the polymerase helps to ....
    keep the two DNA strands separate whilst in the "bubble"
  41. Two types of Proofreading
    • Kinetic Proofreading
    • Nucleolytic Proofreading
  42. Kinetic Proofreading
    • Detects (via stalling) mismatched immediately after phosphodiester bond formation
    • Reverses the catalytic reaction (pyrophosphorolysis)
  43. Nucleolytic Proofreading
    • polymerase backtracks and nuclease activity hydrolyzes bond upstream of mismatched base
    • RNA polymerase makes 10-100x more errors than DNA polymerase
  44. Two ways to terminate transcription in bacteria:
    • 1)Rho-Independent
    • 2)Rho-Dependent
  45. Rho-Independent
    Requires two sequences in the RNA
    • a)Termination sequence in RNA about 15-20 nucleotides from end of RNA.
    • •Creates hairpin structure
    • b)AAA in template strand creates UUU in RNA strand
    • •Hairpin dislodges RNA at AAA/UUU interface
  46. Rho-Dependent
    rut site:
    rho utilization site!Sequence on RNA that recruits rho
  47. Rho-Dependent
    ρ (rho) helicase:
    helicase that travels along RNA transcript
  48. Rho-Dependent
    When ρ encounters rut, will hydrolyze ATP to dislodge transcript and shut down transcription
  49. What does the pin region of the RNA polymerase do?

    B. )
  50. Which type of proofreading is essentially the reverse of the polymerase reaction?
    A.Kinetic Proofreading
    B.Nucleolytic Proofreading
  51. What sequence is NOT essential for rho-independent transcription termination?

    A. )
Card Set
Bio 99 Final Lec 10