Lectures 23 & 24 Flashcards
Do DNA binding domains recruit or block RNA pol
either
What are the 5 steps of eukaryotic transcription cycle
Recruitment/ Assembly
Initiation
Elongation
Termination
Recycling
What are general transcription factors? What are the 6
Universal proteins required to get RNA pol II working
TFIID
TFIIA
TFIIB
TFIIF
TFIIE
TFIIH
ABDEFH
Describe the formation of the preinitiation complex
TFIID containing TBP binds the TATA box
TBP causes DNA to partially unwind
TFIIA binds TFIID and stabilizes the association with DNA
What does TFIIA do
TFIIA binds TFIID and stabilizes the association with DNA
Makes TFIID binds better to DNA
What does TFIIB do
Binds the promoter at the TFIIBRE (recognition element)
By binding TFIIB the potential symmetry of TFIID binding is given directionality
Once TFIIB is bound, RNA pol II can be recruited to the promoter
What does TFIIF do
Stabilizes the binding of pol II to TFIIB
What are the stabillizing TFs
TFII-A stabilizes the binding of TFII-B
TFII-F stabilizes the binding of pol-II to TFII-B
Once the polymerase is bound, it need to form the _______ ________ to start RNA initiation
Transcription bubble
what is the role of the mediator
various sequences and proteins act to turn genes “on” and these signals are all integrated by the mediator complex to bring RNA pol II to the gene when it is needed
The Mediator integrates multiple signals
What is an enhancer? why can they act over long distances? Where are they located?
A sequence which binds a specific transcription factor & recruits/stabilizes mediator to increase the likelihood of transcription
Can act over huge distances due to the organization in the nucleus creating large loops
Can be upstream or downstream but has to be somewhere in the genome
What are insulators? Example?
Keeps loops distinct
Prevents enhancers from activating genes in other loops
CTCF is an example which is very important in vertebrates
Describe the process from pre-initiation to initiation
TFIIE and TFIIH finish up the pre-initiation complex
TFIIE assists forming the open-complex as it stabilizes ssDNA and also increases the activity of TFIIH
what is TFII H
Helicase
using ATP it unwinds DNA creating the open-complex
Also has kinase activity to phosphorylate the tail of RNA pol I
what is abortive initiation
production of very short RNA molecules that get stuck
The proteins bound to promoter sequences are also preventing RNA pol II from moving away from those same sequences
what does RNA pol II struggle with? How can it get past it?
Like it’s prokaryotic counterpart, it struggles with abortive initiation
Phosphorylation of the RNA pol II CTD by TFII-H will allow it to disengage from proteins bound at the promoter and undergo what is called “promoter escape
What is the pol II CTD
Unlike RNA pol I and III, RNA pol II has a C-terminal domain, which is a tail of these heptad repeats which can be phosphorylated
What are the states of phosphorylation during the transcription cycle
Loading onto promoter: CTD is dephosphorylated
Initiation: Phosphorylated at Ser5 by TFIIH
Elongation: Ser5 and Ser2 is phosphorylated
Termination: dephosphorylation of Ser5, Ser 2 is still phosphorylated
how does incorporating the wrong nucleotide affect the polymerase
it can pause/stall the polymerase
Why are there misincorporations
Nucleotides and ribonucleotides can spontaneously created different forms (tautomers) that exist transiently
If the tautomer is integrated while it is in the incorrect form then the RNA polymerase will stall and can’t proceed until the nucleotide is removed
What does TFIIS do
TFIIS fixes the stalled RNA pol II
TFII-S stimulates the backtracking and RNA proofreading activity of pol II to hydrolyze misincorporated rNTPs
Overview of the transcription cycle for RNA pol II
TFIID binds TATA box (in TATA-less promoters it binds the initiator sequence and downstream promoter element)
TFIIA stabilizes TFIID
TFIIB binds the BRE and orients the loading of RNA pol II
TFII-F associated with RNA pol II associates with TFII-B for proper RNA pol II placement
Mediator integrates signals from activators/repressors
TFII-E stabilizes ssDNA (transition from closed to open complex)
TFIIH acts as the helicase and phosphorylates Ser5 of RNA pol II CTD to allow promoter escaped
TFIIS enhances removal of misincorporated nucleotides to prevent RNA pol II stalling
P-TEFb adds Ser-2-P to differentiate initiation from elongation
during elongation the polymerase associates with and carries along RNA modifying/processing proteins
What is epigenetic gene control
a layer of heritable gene expression that lies above the DNA sequence
How is epigenetic control expressed through chromatin/histones
Histones can block access to DNA sequences
Heterochromatin is tightly bound and inaccessible
Euchromatin is loosely bound and accessible
Describe histone tails
tails extending from the core that can be modified to all the overall binding affinity
Can be modified by enzymes
Describe the histone code
the effect of various modifications of the four histones on the chromatin state of gene expression
four types of modification
Their effect extends far beyond simply loosening the association between histones and DNA, and can also recruit activators or inhibitors to the promoter
four types histone modification
Acetylation, methylation, phosphorylation, and ubiquitination
Describe Lysine acetylation
Uses HATs and HDACs
HAT = histone acetyl transferases
HDAC = histone deacetylases
Acetylation removes the positive charge from lysing, reducing the affinity of histones to the DNA backbone
describe the effects of histone acetylation and deacetylation
activation of transcription and transition to euchromatin often involve specific histone acetylations
Repression and transition to heterochromatin often involve deacetylations
Many different HATs and HDACs exist in the nucleus
which TF has HAT activity
TFIID
Which residues can be methylation
Lysine and arginine
Methylation is additive lysine can be trimethylated
Arginine can be dimethylated
T/F Acetylation and methylation are not mutually exclusive
false, can’t be methylated an acetylated. methylation is a way to prevent acetylation
T/F Modification don’t affect other modifications
False
What is the typical effect of methylation
usually silencing but there is some activation
how can the histone code be visualized
ChIP and a series of antibodies that are specific for individual acetylation and methylation
The code can be visualized when overlaid on important gene sequences
