exam 2 (eukaryote transcription) Flashcards
regulation of gene expression is done by
promoters
regulation of transcription is done by
DNA binding proteins
whats the difference between prokaryotic and eukaryotic transcription?
more complicated with multiple RNAPs, more initiation factors, and chromatin
RNAP1 is associated with
rRNA
RNAP2 is associated with
mRNA, IncRNA, miRNA
what are introns
protein non-coding regions in genes
what are exons
protein coding regions in genes
what are the eukaryotic core promoters
BREs: TFIIB response elements
TATA box
Inr: initiator element
DPE: downstream promoter element
how does eukaryotic RNAPii differ from bacterial RNAP
eukaryotic RNAPii holoenzymes can’t bind promoters wihtout help
what occurs with the preinitiation complex (PIC)
RNAPii is recruited to promoters, which contains general transcription factors
what is the TATA-binding protein and its function
subunit of TFIID
binds TATA boxes to initiate PIC formation
(only ~10% of promoters have TATA boxes)
what is TFIID
- contains TBP and 13 associated factors
- contacts Inr and DPE
- structure to support PIC assembly
- integrates regulatory signals
TFIIH function
- helps unwind DNA to form the open complex (acts as helicase)
- it phosphorylates the RNAPii CTD
what is the other promoter on the gene that is upstream of the core promoter
regulatory promoter
whats the difference in the two eukaryotic gene promoters
regulatory promoters: has binding sites for specific activators
core promoters: bounded by GTFs
how is the strength and the contribution of a promoter tested?
reporter assays
common reporters
Luciferase
green fluorescent protein (GFP)
lacZ
what is the role of a reporter assay
bonds to a promoter in a gene, it is transcribed and translated, and is created into a reporter protein
what are enhancers
DNA regulatory element that activates transcription to higher levels, by providing a binding site to be looped in relation to the target promoter gene
for example, if SV40 was added to a eukaryotic gene, what would occur?
transcription would increase 100-fold
at what distance does a enhancer need to be at, and what direction did it move
can occur over a decently far distance, and can work in forward or backwards direction
decsribe enhancer properties
distance independent:
can occur over a decently far distance, 1 kb
orientation independent:
can work in forward or backwards direction
how are complex enhancers created
transcription factors bind to numerous enhancers to form a complex code
how are enhancers and promoters similar
contain specific DNA sequences bound by various proteins
functions of insulators
block enhancer activity
create a boundary between euchromatin and heterochromatin
protein CTCF mediates which function
it is the bridge of the insulator
what are the two domains an activator must have
DNA-binding domain: makes contact with specific DNA sequences in promoters and enhancers
Activation domain: interacts with various components of the transcription machinery
what bonds to both the enhancer and regulatory promoter
DNA binding domain and activation domain, together creating the activator proteins
how is transcriptional activations isolated
protein extract and specific DNA sequence that is bonded to beads are mixed. Non-specific DNA is added to complete any proteins that bind DNA nonspecifically. Salt is added to release proteins specifically bounded to the beads. Pure regulatory protein is released and beaded DNA remains
how are activators used to screen for proteins that interact with one another
2 hybrid assay
expression of the normal activator leads to…
normal transcription of the reporter gene
expression of the bait-DNA binding domain fusion alone leads to
no reporter expression
expression of the prey-activation domain fusion leads to
no reporter expression
expression of the bait and prey fusions (assuming they are 2 interacting proteins) leads to
robust reporter expression
how are activators classified
by the structure of their DNA bonding domains
what kind of bonds do activators and DNA have
hydrogen bonds
ionic bonds
hydrophobic interactions
NOT COVALENT
describe helix-turn-helix (HTH) model
semicircular, fitting in the major groove of DNA complex
helix 3 is hydrophilic
why do most HTH sequence specific DNA binding proteins bond the major groove
more features of the bases are exposed
the HTH recognizes what sequence
5’ AATA 3’
function of zinc fingers (ZFs)
many different types that use cysteine and hstidine residues to coordinate zinc ions and properly fold the domain
describe the ZF of Gal4
a yeast activator that uses 6 cysteines to coordinate 2 zinc ions
Zn2Cys6ZF
what is Gal4
- dimer (must be to operate)
- stabilized by coiled coil
- uses Zn2Cys6ZF to make specific contacts with major groove, spaced 17bp apart
what is Gcn4 and its function
- dimer
- stabilized by coiled coil
- forms scissor-like structure that makes hydrogen bonds with specific bases and ionic bonds with oxygen atoms in the backbone
what is unique about the connection of the mediator complex
there is a bridge between enhancer and regulatory promoter, there is no physical connection
how many subunits does the mediator complex have
25
what is the function of the mediator head
contacts the PIC
whats the function of the mediator middle
contacts the various other factors and is a structural support
what is the function of the mediator tail
contacts TFs bound to the the enhancer
how does mediator of yeast and mammals compare
mammals have a lot more subunits in the mediator
in bacteria how does RNAP get the energy it needs in order to escape the promoter
scrunching
in eukaryotes, how does RNAP get the energy it needs in order to escape the promoter
scrunching, in addition to new mechanisms
what is the largest subunit in the RNAPii? and what does it consist of?
Rpb1
consists of repeats of AA sequence: YSPTSPS
how many of the Rpb1 residues can be phosphorylated
5/7
how is serine 5 phosphorylated? and its purpose
by TFIIH, during initiation
enables RNAPII to break free of the PIC
when are serines 2 and 7 phosphorylated
during elongation
RNAPII is regulated by what factors
NELF and DSIF
RNAPII is released by
P-TEFb
where does RNAP pausing occur
in a window downstream of where the start of transcription begins (Inr and exons)
why does RNAPII pause
balances genes for rapid synchronized activation by preloading RNAPII
describe elongation
RNA strand reads off of the DNA template strand, in the RNAP to match the complementary strand (only t is replaced with u)
Pausing is regulated by
NELF, DSIF, P-TEFb
why does RNAPII stall
physical impediments to progress, could be error correction, not enough energy etc
what is a nucleosomal role in transcription
it prevents it from progressing, by putting up a substantial energy barrier to RNAPII
how does RNAPII get through the nucleosomal DNA
histone variant H2A.Z helps
