Bio 99 Final Lec 11

  1. CTD (C-Terminal Domain)
    • eukaryotic region that is involved in a LOT of things
    • A docking platform for other factors involved in transcription
  2. Pol I: RNA Products and Promoter Elements
    • rRNAs
    • UCE, core sequence
  3. Pol II: RNA Products and Promoter Elements
    • mRNA
    • BRE, TATA box, Inr, DPE
  4. Pol III: RNA Products and Promoter Elements
    • tRNA 
    • Box A, Box B
  5. TATA Box
    • a sequence that contains TATAAA near position -30
    • a common sequence in eukaryotic promoters
    • usually resides in the minor groove of DNA 
    • not all promoters have TATA boxes
  6. TBP-- TATA-binding protein
    • binds to the TATA box
    • fits onto the TATA box like a saddle
    • involved in transcription initiation of almost all genes (even ones that lac a TATA box)
  7. TAFs-- TBP-associated factors
    help to recruit TBP to the promoter
  8. Pol I Promoter
    • transcribes most rRNAs
    • 80% of transcription is Pol I
    • contains Core Sequence and Upstream Control Element (UCE)
    • UBF(upstream binding factor) binds to core sequence and UCE
    • SL1 (contains TBP and 3 TAFs) binds UBFs and helps recruit RNA Pol I
  9. Pol III Promoters
    • mainly for tRNA, but also 5S rRNA and some specialized RNAs
    • All their targets are short (less than 300 nucleotides) and untranslated
    • Part of their promoter region is within the gene(downstream of the start
    • site)
    • All Pol III promoters require TBP binding to a TATA box, with a complex
    • called TFIIIB (“TF3B”)
    • All Pol III promoters have TFIIIC (“TF3C”) binding to boxes downstream of the start site
    • TFIIIC gets kicked off by Pol III once transcription begins
  10. Pol II Promoters: Promoter Sequences
    • Inr – Initiator, at the start site
    • DPE – Downstream promoter element
    • TATA box – binds TBF
    • BRE – TFIIB recognition element, binds TFIIB
  11. Pol II Promoters: Transcription Factors
    • TBP – Binds TATA Box
    • TFIIB – Binds BRE
    • TFIID – massive complex with many TAFs, and TBP
  12. Mediator Complex
    • Massive complex of proteins involved in bringing multiple transcription factors that bind to distal regions in close proximity to initiation complex
    • Think of it as scaffolding
  13. Which transcription factor is used by all three Pol promoters?

    A. )
  14. What feature of Pol I promoters makes it similar to Bacterial promoters?

    C. )
  15. What is a similarity between Pol II and Pol III promoters?

    the start site
    D. Both use BRE
    C. )
  16. ______ complex converts to ____ complex similar to closed to open configurations in bacterial initiation
    • preinitiation
    • initiation
  17. ________ of CTD region involved in disenganging from the promoter
  18. Torpedo Model for Pol II Termination (4 Steps)
    • 1.) termination sequence AAUAAA signals for a complex to cleave mRNA, the 3' end is further processed
    • 2.) even though transcript is finished, Pol II continues to transcribe
    • 3.) Xrn2, an exonuclease, begins to chew up the leftover RNA
    • 4.) when it catched up to Pol II, it removes the transcript and ends transcription, similar to Rho-dependent termination in bacteria
  19. In which of the following steps of transcription is the C-terminal
    domain of RNA polymerase II first phosphorylated?

    D. )
  20. Which of the following is NOT true about the role of Mediator
    complex in transcription initiation?

  21. Northern Blot
    • Run whole RNA preps on a gel, transfer to a
    • membrane, probe membrane with a radiolabeled DNA probe that
    • matches your gene of interest
  22. RT-PCR
    • Convert RNA to cDNA, PCR using primers within your
    • gene of interest
  23. qRT-PCR
    • same as RT-PCR, but use quantitative PCR machine
    • to measure transcript levels
  24. RNA sequencing
    convert RNA to cDNA. Next gen sequencing
  25. gel electrophoresis
    • Purpose – to separate and visualize DNA/RNA/protein molecules based on size
    • Gel – Made out of agarose (for DNA/RNA). Porous, which allows DNA/RNA to
    • pass through it.
    • DNA/RNA is loaded into holes in the gel (wells). A blue loading buffer weighs it
    • down and keeps the DNA/RNA from floating out of the well.
    • Gel is placed in buffer in a chamber and an electrical gradient is applied
    • • Smaller molecules move faster through the gel than larger molecules
  26. probes
    • Fragments of single-stranded DNA or RNA will attach to complementary
    • sequences (e.g. template strand to coding strand)
    • Hybridization – the attachment of a single-stranded fragment of DNA (or
    • RNA) to a complementary sequence (not it’s opposite strand).
    • Probes can be created from fragments of known DNA and then labeled.
    • When added to a Southern or Northern blot, the probe will hybridize to any
    • DNA/RNA that has complementary sequence (even if double-stranded)
  27. Northern Blot – a method to analyze RNA
    amounts and sizes
    • 1) RNA extract is run through an agarose gel
    • 2) The RNA is then transferred from the gel to a
    • nitrocellulose membrane
    • 3) The membrane can be probed with a DNA
    • probe that is radiolabeled
    • Probe will bind to complementary RNA
    • sequences
    • 4) The membrane is imaged with x-ray film to
    • detect the presence of the radiolabeled probe
    • The intensity of the band is proportional
    • to how much RNA is present
  28. PCR – Polymerase Chain Reaction
    What is it for?
    amplify short regions of DNA in order to visualize it
  29. Describe PCR
    • Amplification of a region of DNA
    • using 2 primers
    • Amplified region (aka amplicon) is
    • in between the 2 primers
    • Each cycle of amplification
    • “doubles” the amount of PCR
    • product
  30. RT-PCR
    Reverse Transcriptase PCR
    What is it for?
    • A way to convert RNA into DNA in
    • order to amplify it via PCR.
    • A way to visualize RNA transcripts
    • without using a Northern blot
  31. RT-PCR
    Reverse Transcriptase PCR
    How you do it:
    • 1) treat RNA with reverse
    • transcriptase enzyme to convert to
    • cDNA (complementary DNA).
    • 2) Use cDNA in a PCR reaction.
  32. Quantitative PCR (or qPCR)
    What it is:
    • A method to quantify the amount of PCR product, and
    • thereby the amount of starting DNA
  33. Quantitative PCR (or qPCR)
    How it does it:
    • : A dye that binds to DNA is used in a PCR reaction. The
    • dye gives off a fluorescent signal which is proportional to the amount
    • of DNA in the tube. A special PCR machine reads the signal after each
    • PCR cycle
  34. SYBR green
    • Binds only to double stranded DNA
    • (dsDNA)
    • When bound, emits fluorescent signal
    • Fluorescent signal is proportional to
    • amount of double stranded DNA
    • • In PCR reaction, can measure signal
    • after each cycle
    • Signal should double each cycle as amount of dsDNA doubles
  35. Quantitative PCR (or qPCR)
    3 phases
    • In the few several cycles, there is not enough PCR product (i.e. dsDNA) to be detected.
    • After about 15 cycles, there is enough PCR product to be detected, and each cycle the signal increases by about 2 fold. This is known as the “exponential phase”.
    • After around 30 cycles, the reagents (dNTPs, primers, polymerase) get used up, and the signal plateaus.
    • Plotting the signal after each cycle produces an amplification curve
  36. Quantitative PCR (or qPCR)
    Analysis (comparing multiple samples)
    • Image Upload 1
    • An arbitrary signal threshold is set (dashed line) at the exponential phase
    • The cycle at which each sample crosses the threshold (Ct) is determined (doesn’t have to be a whole number)
    • The difference in Ct values (DCt) is used to estimate the difference in concentration of the samples. For example, a one cycle difference in Ct between two samples is equivalent to a two-fold difference in concentration
  37. qPCR can be used to measure gene expression if RNA is converted to cDNA, then analyzed via qPCR. This is called ....
  38. In order to be quantitative, you need two controls:
    1.) Loading control
    2.) Population control
    (define each)
    • 1.) primers for a gene whose expression is likely to be the same in every cell (i.e. housekeeping gene). Used to normalize all your samples to one another
    • 2.) A sample to compare your sample of interest to. For example, wild-type versus mutant, or different brain regions, or untreated versus treated.
  39. Flowchart of the 4 PCR methods
    Image Upload 2
  40. NextGen Sequencing Aka High Throughput Sequencing
    What it is:
    A method to sequence a massive amount of DNA at once, ~120 Gb (human genome is ~3 Gb)
  41. NextGen Sequencing
    Aka High Throughput Sequencing
    How it does it:
    Sequences short regions of DNA (~150bp), but a massive number (~400 million “reads”), then assembles them via computer
  42. NextGen Sequencing
    Aka High Throughput Sequencing
    Overview (massively simplified):
    • DNA chopped into small fragments (few hundred bps)
    • DNA fragments run through a microfluidic system that deposits them into tiny nanowells, essentially one single-stranded DNA fragment per nanowell
    • DNA polymerase reads each single strand and copies it
    • Each dNTP emits a different wavelength of fluorescent light as it is added
    • A camera captures the light emitted by each nanowell, knowing whether it is A, T, G, or C
    • Around 150 bases per well can be read this way, these are called “reads”
Card Set
Bio 99 Final Lec 11
Eukaryotic Transcription