Central Dogma Lecture 13 Transcription III

Question 1

What is CDT?

Answer 1

-Mammalian Rpb1 C-terminal domain (CDT)
-Has 52 repeats of PTSPSYS

Question 2

Phosphorylation and dephosphorylation of CTD

Answer 2

CDT can be phosphoyrlated by CTD kinases and dephosphoyrlated by CTD phosphatases

Question 3

CDT and initiation of transcription

Answer 3

-RNAP II initiates transcription only when the CTD is unphosphorylated
-Commences elongation only after phosphoylation
-Phosphorylation launches transcription

Question 4

Structure of RNAP II

Answer 4

-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

Question 5

How does RNAP differentiate NTPs from dNTPs?

Answer 5

-Specificity for NTP over dNTP because enzyme recognizes incoming ribose
-Enzyme only accepts NTPs that form a Watson-Crick base pair with the template

Question 6

α-Amaanitin inhibits RNAP II and III

Answer 6

-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

Question 7

Promoter regulation in bacteria vs eukaryotes

Answer 7

-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

Question 8

Promoter basics in eukaryotes

Answer 8

-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

Question 9

TATA box

Answer 9

-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

Question 10

Mechanisms for recognition of DNA sequences that direct transcription in bacteria and eukaryotes

Answer 10

-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

Question 11

What are the general/basal transcription factors which are used to form PIC?

Answer 11

-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

Question 12

What are the steps for assembly of the pre-initiation complex (PIC)

Answer 12

-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

Question 13

What activity does TFIIH have?

Answer 13

-Helicase activity which uses ATP to melt the promoter to initate transcripion at basal rate
-Phosphorylates RNAP II CTD, necessary for elongation to start

Question 14

TATA binding protein and how it binds TATA box

Structural changes

Answer 14

-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

Question 15

What RNAPs are in eukaryotes, what do they do, and where are they localized?

Answer 15

-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

Question 16

How do mitochondrial and chloroplast RNAPs compare to eukaryotic?

Answer 16

-Distinct mitochondrial and chloroplast RNAPs
-Simpler single subunit RNAPs
-Reminiscent of bacteriophage RNAPs

Question 17

What are eukaryotic RNAPs made up of?

Answer 17

-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

Question 18

What is the nucleolus?

Answer 18

Dense granular bodies in the nucleus that contain the ribosomal genes

Question 19

Do eukaryotic RNAPs independently bind the promoter?

Answer 19

-No
-They are recruited to target promoters by transcription factors (TF)

Question 20

What is Rpb1?

Answer 20

-RNAP II eukaryotic holomolg to bacteria β′ subunit

Question 21

Structure of TBP-TATA-box complex

Answer 21

-Asymmetric
-Crucial for specifying a unique start site and ensuring that transcription proceeds unidirectionally

Question 22

Composition of upstream transcription factors

Domains

Answer 22

-2 domains:
-DNA-binding domain which binds the target DNA sequence
-Transactivation domain, which activates transcription by binding PIC

Question 23

What is the Mediator?

Answer 23

-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

Question 24

What are enhancers?

Answer 24

-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

Question 25

How do enhancers participate in transcription (initiation) if they are not near promoter?

Answer 25

-DNA loops around so that TF can contact enhancer, promoter, and RNAP II

Question 26

What are enhancers required for and what are they associated with?

Answer 26

-Required for full activity of cognate promoter
-Enhancers are associated with genes that are selectively expressed in specific tissues
-Mediate much of the selective gene expression in eukaryotes

Question 27

What happens to PIC when it has been used to initiate transcription for an RNAP?

Answer 27

-Formation of a stable PIC controls transcription by loading multiple RNAP IIs
-Once you build PIC, it can load RNAP IIs over and over to create transcriptional bursts
-RNAP II transcribes genes at a steady ~50 nt/sec

Question 28

Reverse transcriptase enzyme

Answer 28

-RNA-dependent DNA polymerase
-Looks like a hand, not a claw
-Heterodimer
-Two cupped hands
-Only one active site
-No exonuclease domain
-Error prone
-Poor processivity
-RNAse H domain

Question 29

What is the RNAse H domain?

Answer 29

-Degrades duplexed RNA
-on one part of heterodimer
-Endonuclease
-Active site is 17-18 bp behind 3’ end

Question 30

What uses reverse transcription?

Answer 30

HIV uses a host tRNA as a primer to start DNA synthesis

Question 31

Reverse-transcription steps

Answer 31

-tRNA ‘primer’ binds RNA
-RT makes DNA:RNA hybrid
-Rnase H chews up RNA
-RT uses ssDNA to make dsDNA

Question 32

How is recombinant RT useful?

Answer 32

Useful in the lab to make cDNA out of mRNAs for next-gen sequencing

Question 33

Inhibiting RT

Answer 33

-AZT is incorporated into DNA instead of thymidine
-Cell adds a triphosphate
-Presence of 3’ azide means growing chain is terminated

Question 34

Why is AZT useful for inhibiting RT?

Answer 34

-RT has higher affinity for AZT compared to dTTP
-Human DNA pol has lower affinity for AZT compared to dTTP