Lecture 2 midterm 3

• CTD (C-Terminal Domain)


  Eukaryotic region that is involved in a LOT of things. A docking platform for other factors involved in transcription.
• 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 lack a TATA box)
• 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
• TAFs (TBP-associated factors)


  Help to recruit TBP to the promoter
• Pol I


  Transcribes most rRNAs, 80% of transcription is Pol I
• UBF (upstream binding factor)


  Binds to core sequence and UCE
• Pol I Promoter


  Contains Core sequence and Upstream control element (UCE)

  
• SL1 Complex


  Contains TBP and 3 TAFs, connects UBF and helps to recruit RNA Pol I
• 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)
• TFIIIB ("TF3B")


  All Pol III promoters require TBP binding to a TATA box, with this complex
• TFIIIC ("TF3C")


  All Pol III promoters have this binding to boxes downstream of the start site, gets kicked off by Pol III once transcription begins
• Pol II Promoter Sequences

  • Inr – Initiator, at the start site
  • DPE – Downstream promoter element
  • TATA box – binds TBP
  • BRE – TFIIB Recognition Element, binds TFIIB

  
• Transcription Factors


  • TBP – Binds TATA Box
  • TFIIB – Binds BRE
  • TFIID – massive complex with many TAFs, and TBP
• Mediator Complex


  Massive complex of proteins involved in bringing multiple transcription factors that bind to distal regions in close proximity to initiation complex, acts to speed up assembly
• Preinitiation complex converts to initiation complex, much like closed to open configurations in bacterial initiation, leads to loading of DNA into entry channel, melting of DNA and formation of transcription bubble

  
• Phosphorylation of CTD region DURING INITIATION is involved in disengaging from the promoter
• Elongation similar to bacteria, but facilitated by elongation factors
• During termination, CTD is dephosphorylated
• Torpedo model for Pol II termination

  • 1) Termination sequence AAUAAA signals for a complex (CPSF) 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 catches up to Pol II, it removes the transcript and ends transcription
• 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
• RT-PCR


  Convert RNA to cDNA, PCR using primers within your gene of interest
• qRT-PCR


  Same as RT-PCR, but use quantitative PCR machine to measure transcript levels
• RNA sequencing


  Convert RNA to cDNA, Next gen sequencing
• 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), Smaller molecules move faster through the gel than larger molecules

  
• Probes


  Fragments of single-stranded DNA will attach to complementary sequences, Hybridization – the attachment of a single-stranded fragment of DNA (or RNA) to a complementary sequence, Probes can be created from fragments of known DNA and then labeled, When added to a Northern blot, the probe will hybridize to any RNA that has complementary sequence (even if double-stranded)
• 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, 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

  
• Northern blots are used to quantify the amount of RNA transcripts in a group of cells
• Radioactive DNA


  Northern Blot
• Northern Blot


  A method to analyze RNA amounts and sizes
• Northern Blot 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

  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
• Images of Northern blots are always shown with the wells on top, so the higher bands are larger than the lower bands
• PCR


  To amplify short regions of DNA in order to visualize it
• PCR


  1. Amplification of a region of DNA using 2 primers

  2. Amplified region (aka amplicon) is in between the 2 primers

  3. Each cycle of amplification "doubles" the amount of PCR product
• RT-PCR (Reverse Transcriptase PCR)


  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.
• RT-PCR


  1. Treat RNA with reverse transcriptase enzyme to convert to cDNA (complementary DNA)

  2. Use cDNA in a PCR reaction

  
• 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

  
• Quantitative PCR (or qPCR)

  A method to quantify the amount of PCR product, and thereby the amount of starting DNA
• Quantitative PCR (or qPCR)

  1. A dye that binds to DNA is used in a PCR reaction

  2. The dye gives off a fluorescent signal which is proportional to the amount of DNA in the tube

  3. A special PCR machine reads the signal after each PCR cycle

  
• Plotting the signal after each cycle produces an amplification curve