Molecular Chapter 20

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Molecular Chapter 20
2012-03-05 13:06:33
Eukaryotic Transcription

Chapter 20
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  1. Describe the general properties of promoters and enhancers.
    • 1. Promoters typically extend upstream from the initiation site and contain several short (~10bp) sequences that bind transcription factors
    • 2. An enhancer with may be located even further upstream of initiation relative to a promoter and also has sequences that bind transcription factors
  2. Describe the general characteristics of RNA pol II.
    • 1. Largest subunit has ~50 CTD repeats
    • 2. CTD can be highly phosophorylated on serine and threonine residues
    • 3. CTD is involved in regulation of initiation, elongation, mRNA processing, and export of mRNA to cytoplasm
    • 4. The RNA pols in mitochondria and chloroplasts resemble bacterial RNA pol
  3. What is the function of RNA pol I?
    • 1. It only transcribes 28S and 18S rRNAs
    • 2. The rRNAs are produced as a primary transcript and later cleaved into separate rRNAs
  4. Describe the organization of the promoter and the events involved in initiation of transcription by RNA pol I.
    • 1. UPE located at -180 to -107 and a core promoter region spanning the start point (-45 to +20)
    • 2. UBF binds the UPE, increasing the binding efficiency of SL1
    • 3. Transcription factor SL1 positions RNA pol 1 at the start point via its TBP domain.
  5. What is the function of UBF?
    • 1. Binds DNA minor grove and loops DNA bringing core promoter region into proximity
    • 2. Interacts with SL1 to help it bind to core promoter region
  6. What are the types of promoters for RNA pol III.
    • 1. Internal promoters (5S rRNA and tRNA) - promoters lie downstream of start point
    • 2. Upstream promoters (snRNA) - promoters lie upstream of start point
  7. Summarize the stages of reaction at type 2 internal promoters used for tRNA genes.
    • 1. Assembly factor TFIIIC binds to boxA and boxB, which
    • 2. Recruits positioning factor TFIIIB, which has TBP and binds TATA region, which
    • 3. Recruits RNA pol III
  8. Summarize the stages of reaction at type 1 internal promoters used for tRNA genes.
    • 1. Assembly factors TFIIIA and TFIIIC bind to boxA and boxC respectively
    • 2. Recruits positioning factor TFIIIB which has TBP and to bind TATA region
    • 3. Recruits RNA pol III
  9. Describe the construction of pol II with 3 of its most common promoter elements.
    • 1. TATA box (TATAA) ~25 bps upstream of Inr
    • 2. Initiator (-3 to +5) has pyrimidines (Y) surrounding the CA at the startpoint
    • 3. The downstream promoter element (DPE) is at +28 to +32 for the ~50% TATA-less promoters
    • 4. Typical core promoters consists of a TATA box + Inr or Inr + DPE
  10. What is the universal factor for each type of RNA pol?
    TBP - TATA binding protein
  11. TBP is a part of what TF that recognizes RNA pol II?
  12. Describe the binding of TBP to DNA
    • 1. C-Terminus bind to the DNA
    • 2. TBP forms a saddle over the minor grove
    • 3. Bends the TATA box ~80 degrees towards the major groove allowing TFs and RNA pol to form tighter associations to DNA
    • 4. N-Terminus free to interact with other proteins
  13. What are the three possible chromatin transcription stages?
    • 1. Inactive gene - closed chromatin
    • 2. Poised gene - potentially active gene; open chromatin; basal apparatus assembled; gene needs 2nd signal to start transcription
    • 3. Transcribed gene - open chromatin; active transcription
  14. Describe the steps in activating a TATA box-containing promoter
    • 1. TFIID binds at TATA box - TFIID also binds Inr and DBE
    • 2. TFIIA joins complex causing TFIID to bind further upstream and activates TBP
    • 3. TFIIB binds minor groove downstream of TATA box next to TBP and major groove upstream of TATA box; determines polarity of RNA pol II
    • 4. TFIIF binds complex with RNA pol II along with TFIIE and TFIIH to complete the initiation complex
    • 5. TFIIH mediates promoter melting via ATP-dependent helicase action
    • 6. All factors except E, F, and H are released from RNA pol II
    • 7. Once elongation occurs, TFIIS binds to prevent inappropriate pausing and enzymes and factors bind to the CTD
  15. Describe the interaction of TFIIB with RNA pol II.
    • 1. N-terminus influences switch from abortive initiation to promoter escape
    • 2. Inserts elongated finger into active center of RNA pol II
    • 3. C-terminus orients DNA via RNA pol II/TFIID interaction
    • 4. Determines DNA path by aligning TFIIE, TFIIF, and TFIIH
  16. What is the function of TFIIF?
    • 1. Helicase subunit that melts DNA promoter region
    • 2. Sigma factor-like subunit brings RNA pol II to initiation complex
  17. What is promoter clearance?
    • 1. The final step in RNA pol II releasing from promoter region
    • 2. Key regulatory step in poised gene/active gene transcription
    • 3. Controlled by enhancers
    • 4. TF bind to coactivators that bind to enhancers
    • 5. Mediator is one of the most common coactivators
  18. What is the function of dynamic phosphorylation of CTD tail of RNA pol II in elongation?
    • 1. Mediator specifically interacts with CTD of unphosphorylated RNA pol II
    • 2. This interaction is part of TF and promoter binding
    • 3. Phospohorylation disrupts this interation causing release.
    • 4. Phophorylated sites are recognized by elongation enzymes
    • 5. E.g. capping, splicing, and 3' end processing enzymes
  19. How is chromatin remodeled during transcription
    • 1. RNA pol II has enzyme/chaperone that removes histone dimer
    • 2. Hexamer easier to displace/reassemble than octamer
    • 3. DNA template strand repaired during transcription
    • 4. non-template strand reparied with bulk DNA
    • 5. TFIIH - 2 helicase subunits (XPB, XPD) that function in initiation and repair
  20. Describe the enhancers CAAT box and GC box
    • CAAT box
    • 1. Upstream enhancer at -80
    • 2. Determines promoter efficiency but not specificity
    • 3. GC box at -90 GGGCGG
  21. compare and contrast enhancers and promoters
    • 1. Position relative to promoter need not be fixed
    • 2. can function in either orientation relative to promoter
    • 3. Both are short sequences that bind TFs
  22. Describe the two type of TFs (activators and repressors) interaction with enahncers
    • 1. Direct interaction w/basal transcription machinery or via coactivators (Mediator)
    • 2. Chromatin remodeling - recruit modification enzymes/remodeling complexes or by bending DNA
  23. How do enhancers increase gene transcription?
    • 1. Increase [TF] near promoter
    • 2. cis-acting - DNA must loop if enhancer is far from promoter
    • 3. enhancer is limited by insulator sequences or protein-protein specificity
  24. How is gene expression associated with Demethylation
    • 1. Methylation at a promoter may inhibit transcription
    • 2. Methylation at RNA pol II promoters occurs at CG doublets (CCGG)
    • 3. Methylation can be examined using restriction enzymes - they differ at methylated sites
    • 4. In the chicken alpha-globulin gene undermethylation is present from ~500 bps upstream of the first gene to ~500 bp downstream of the second gene - these genes are being actively transcribed suggesting that methyl groups are associated with the ability of a gene to be transcribed.
  25. How do CpG Islands serve as regulatory targets
    • 1. CpG doublets occur at only 20% of the expected frequency given the proportion of G-C bps
    • 2. CpG Islands - genomic regions where CpG exceeds general frequency by 10X
    • 3. CpG Islands - generally unmethylated
    • 4. Between human and mouse genomes - ~10,000 CpG islands exhibit synteny and undergo chromatin changes consistent w/transcription
    • 5. Genes with extensive CpG islands - expressed constitutively (housekeeping gene)
  26. How does methylation of a CpG island affect transcription?
    • 1. Methylation of a binding site for TFs can prevent binding
    • 2. Methylation may cause specific repressors to bind
    • 3. MeCP1 and 2 - methyl binding repressors
    • 4. MeCP2 - binds directly to methyl group and
    • 5. MeCP2 - can recruit Sin3 repressor complex which has histone deacetylase activity
  27. What are the three changes necessary for transcription
    • 1. Hypersensitive site is established near promoter
    • 2. Chromatin of a domain, including transcribed region, becomes more sinsitive to DNase I
    • 3. DNA of the same region us undermethylated