Central Dogma Lecture 13 Transcription III Flashcards
(34 cards)
What is CDT?
-Mammalian Rpb1 C-terminal domain (CDT)
-Has 52 repeats of PTSPSYS
Phosphorylation and dephosphorylation of CTD
CDT can be phosphoyrlated by CTD kinases and dephosphoyrlated by CTD phosphatases
CDT and initiation of transcription
-RNAP II initiates transcription only when the CTD is unphosphorylated
-Commences elongation only after phosphoylation
-Phosphorylation launches transcription
Structure of RNAP II
-Claw-like shape
-Rbp1 and Rbp2 serve as a “clamp” to trap DNA in the main channel, conferring high processivity
-Active site contained on Rbp1, contains 2 Mg2+ ion
-Funnel region where NTPs enter the enzyme to gain access to the active site
-Rudder which separates the short RNA-DNA hybrid, allowing dsDNA to reform
-Trigger loop of Rpb1 swings in when a correct NTP binds to form an extensive hydrogen bonded network involving both the NTP and RNAP
-Links catalysis with fidelity
How does RNAP differentiate NTPs from dNTPs?
-Specificity for NTP over dNTP because enzyme recognizes incoming ribose
-Enzyme only accepts NTPs that form a Watson-Crick base pair with the template
α-Amaanitin inhibits RNAP II and III
-Comes from death cap mushroom
-Interacts with bridge helix and trigger loop
-Interferes with conformational change of the trigger loop postulated to promote catalysis
-Slows RNAP II and II rate of synthesis to a few nucleotides per minute
-Death occurs after several days due to slow turnover of eukaryotic mRNAs and protein
Promoter regulation in bacteria vs eukaryotes
-In bacteria, most promoters are on and regulated by factors that keep the off
-In eukaryotes, most promoters are off and regulated by factors that turn them on
Promoter basics in eukaryotes
-In eukaryotes, most promoters are off and regulated by factors that turn them on
-RNAP I and II promoters are generally 5’ of gene, while RNAP III promoters are within transcribed regions
-Have several important features, including TATA box and initiator element
TATA box
-The most commonly recognized promoter element for genes transcribed by RNA polymerase II
-Consensus sequence -25 to -30 upstream of TSS (transcription start site)
-Resembles -10 region of prokaryotic promoters
Mechanisms for recognition of DNA sequences that direct transcription in bacteria and eukaryotes
-10 and -35 sequences in bacterial promoters are binding sites for RNAP holoenzyme
-TATA, CAAT, GC boxes, and more in eukaryotic promoters are recognized by proteins other than RNA polymerase itself
What are the general/basal transcription factors which are used to form PIC?
-Eukaryotic RNA polymerase has little ability to bind its promoters
-RNA polymerase II requires six general/basal transcription factors:
-TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
-Combine to form a pre-initiation complex (PIC) at the core promoter
What are the steps for assembly of the pre-initiation complex (PIC)
-TATA binding protein (TBP) binds to the TATA box to identify the TSS
-TBP is joined by ~16 TAFs (TBP-associated factors) to form TFIID
-TFIIA and TFIIB bind and stabilize the complex
-TFIIF binds and recruits RNAP II
-TFIIE and TFIIH join to form the PIC
-TFIIH helicase activity uses ATP to melt the promoter to initate transcription at basal rate
-TFIIH phosphorylates RNAPII CTD
What activity does TFIIH have?
-Helicase activity which uses ATP to melt the promoter to initate transcripion at basal rate
-Phosphorylates RNAP II CTD, necessary for elongation to start
TATA binding protein and how it binds TATA box
Structural changes
-TBP binds 10^5 times as tightly to the TATA box as to nonconsensus sequences
-Kd of the TBP-TATA_box complex is approximately 1 nM
-TATA box of DNA binds to the concave surface of TBP, inducing large conformational changes in the bound DNA
-Flexibility of AT-rich sequences is exploited here in bending the DNA
What RNAPs are in eukaryotes, what do they do, and where are they localized?
-RNA polymerase I, aka Pol I
-Synthesizes most rRNA precursors
-Localized in the nucleolus
-RNA polymerase II, aka Pol II
-Synthesizes mRNA precursors
-Localized in the nucleus
-RNA polymerase IIII, aka Pol III
-Synthesizes tRNA, precursors of 5S rRNA, and other small RNAs
-Localized in the nucleus
How do mitochondrial and chloroplast RNAPs compare to eukaryotic?
-Distinct mitochondrial and chloroplast RNAPs
-Simpler single subunit RNAPs
-Reminiscent of bacteriophage RNAPs
What are eukaryotic RNAPs made up of?
-2 large subunits which are homologs of β and β (Rbp1 in RNAP II)
-Up to 12 small subunits
-2 homologs of α
-1 homolog of ω
-5 of the 12 subunits are identical in all 3 eukaryotic RNAPs
What is the nucleolus?
Dense granular bodies in the nucleus that contain the ribosomal genes
Do eukaryotic RNAPs independently bind the promoter?
-No
-They are recruited to target promoters by transcription factors (TF)
What is Rpb1?
-RNAP II eukaryotic holomolg to bacteria β′ subunit
Structure of TBP-TATA-box complex
-Asymmetric
-Crucial for specifying a unique start site and ensuring that transcription proceeds unidirectionally
Composition of upstream transcription factors
Domains
-2 domains:
-DNA-binding domain which binds the target DNA sequence
-Transactivation domain, which activates transcription by binding PIC
What is the Mediator?
-Bridges DNA-bound upstream TFs and RNAP II, and stabilized PIC
-Binds to unphosphorylated CTD of RNAP II
-Upstream TFs cannot stimulate transcription of a reconstituted PIC without mediator
What are enhancers?
-Transcriptional control elements that do not have fixed positions relative to their corresponding transcribed sequences
-Can be upstream, downstream, or even in the middle of a transcribed gene
-Recognized by specific TFs that stimulate RNAP II to bind to corresponding promoter, though this can be indirect
-Activators/repressors bind to them
