#26 Gene Expression Flashcards
Sites for Gene Expression Control
transcription, pre-mRNA processing, nuclear export, translation, and in some case prtoein activation (phosphorylation)
How do you idetinfy protein expressio levels with 2D polyacrylamide gel electrophoresis?
spearates proteins based on size and charge
vertical axis by weight (heavy on top) and charge on horizonal axis (acidic on left, baisc on right)
What shape is the starte site on the humanenome?
target adopts a very regulat shape B-form DNA
What is the B-form DNA grooves?
Major groove is board and accessible to protein recognition
Minor groove is shallow but still accessible
What is the most distinctive accessible hydrophoic contact?
5’ methyl group of thymine
Why is site-specific recognition challenign in the minor groove?
b/c the accessible features in teh this groove do not differentiate btween A, C, U, and G
The only distinctive H bond acceptor is the distinctive feature of 2’exocylci amine of Guanosine
yep
IT is easy to districminate a bp containing a G form those withotu a G
what is a method to identify protein expressino levels?
perform 2D polyarcrylamide gel electrophoresis that separtes proteins on teh basis of size and charge
How do proteins reach and contact expsed exocyslic functional gorups in major and minot groove of B-form DNA?
they can’t, they use secondary strucutral elemtsn that dock into these grooves
What kind of of secondary structural elemtns dock inot the major groove of DNA?
alpha helix is mst common
Beta hairpins and protein loops are also observed
What do side chains of Asn or Gln recognize?
N6 and N7 of adenosine bases and si
What do side chains of arg reognize?
O6 and N7 of guanin bases
What are helix-turn-helix motifs?
common preotin-DNA recognition motif found in prokaryotes and bacteriophage
Are helix-turn helix motifs homodimers or heterodimers?
homodimers
What is the 1st and 2nd helix
1st helix lies outside of DNA moand turn oritents the second helix in the oppsite direction
Second helix is calle the RECOGNITION HELIX b/c it docks inot he major groove of a B-form helix
PALINDROME
What do eukaryotic TFs emply to dock into major groove of DNA
alpha helices
What are examples of of alha helices used to dock inot major grove of DNA
- Homeodomains
- Zinc Fingers
- Leucine Zippers
What are homeodomains
consist of 3 consecutive alpha helices, with the 3rd one docking into the major groove
What are zinc fingers?
DNA bidnng proteins that contain one or more zinkc atoms
most common DNA bindin domains in euk, 3% of hman gnome
chas beta hairpin and recognition alpha helix that is held together by a covalently bound zinc atom
What are leucine zippers
chopsticks
What are operons?
bacterial genes orgnized into operons that contain a group of genes that carry out a related funciton
what is trp operon?
negative feedback
5 genes in trop operon that cod for 5 enzymes needed to syntehsize amino acid tryptophan
What are the 5 genes needed for trp operon?
Tryp A, B ,C, D, E
what happens when trp concentration is high?
trp binds to trp repressor protein –> recognition helices on trp repressor allows complex to bind to operator site –> bidning of trp repressor complex prevents RNA polymease binding and transcription
at low concnetration of tryp
trp dissociats from repressor to trigger conformatino change that prevents recogntion helices from simultatnusly dockign to major groove of target DNA
does repressor binding turns gene on or off?
OFF
What is positive vs negative feedback repressor bindign
positive feedback- high ligant concentrion drives ligan bnidng to repressor –> repressor dissociates from operator? –> turns genes ON
negative feedback–> high ligant concentraiton drives ligant bindign to repressor –>repressor binds to operator site to sterically blcok RNA polymerase and transcrition_–> turns genes OFF
Activator binding turns genes
ON
activtors are DNA binding roteins that bind to DNA oeprator sites to recrut DNA polymerase
positive feedback vs negative feedback with activator
negative feedback–> high ligand concentraiton drives ligant bidnign to activator –> activator does not bid to operator site –> RNA polymerase is not recruited –> gene is OFF
positive feedback –> high ligan concentration dries liggand binding to actioro –> binds to operator site to recruit RNA polymears and tuen genes ON
lac operon
regulated by both activator and repressor
lac operon codes for protein required to transprot lactose into cell and metbolize disaccharide
What is the catabolic activator protein (CAP) on lac operon
enables bacteria to use non-glucose energy source like lactose
lac repressor is activated when …is scarce and ….is abundant
glucose is scarce….lactose is abundant
lac operon: high glucose, high lactose
operon OFF
presence of glucose leads to low cAMP concentration –> free CAP , doesnt bind to DNA, not active
lactose increases concentration of isomer of lactose allolactose –> binds to lac repressor –> cuases repressor to dissociate from its operator binding site –> RNA poly is not stericlaly locked
but CAP activator off =–> no RNA poly recruited
high glucose, low lactose
CAP off –> no RNA poly recruit
allolactose low –> repressor blocks poly site
low glucose, low lactose
CAP off
repreossr blocks
low glucose, high lactose
CAP binds to DNA
lac repressor dissociated form operator binding site
ONNNN
how can activation occur at a disntance?
enhancer
