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
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