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
    phosphorylation
  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?





    complex.
    B.
  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
    Steps:
    • 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 ....
    qRT-PCR
  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”
Author
jocelyn0399
ID
347324
Card Set
Bio 99 Final Lec 11
Description
Eukaryotic Transcription
Updated