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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
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
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
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.
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
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
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
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
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
What is the universal factor for each type of RNA pol?
TBP - TATA binding protein
TBP is a part of what TF that recognizes RNA pol II?
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
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
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
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
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
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
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
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
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
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
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
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
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.
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)
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
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