116 Lecture Test 3
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116 Lecture Test 3
test lecture 116 molecular genetics
Flash cards on lecture test 3
Experimental procedure to figure out different rnap's?
Ion exchange chromatography
RNA produced by Rnap1?
large RNA precursors
Rna produced by rnap 2?
mRNA's and snRNAS and miRNAS
RNA produced by rnap3?
tRNA's, U6, 5sRNA precursor etc...
Which substance to distinguish between polymerases?
What does alpha amantin do and which polymerases are sensitive?
inhibits RNA synthesis.
rnap2 most sensitive
rnap3 least sensitive
rnap1 not sensitive at all
contains CTD; binds DNA involved in start site selection
contains active site, start site selection, elongation rate
Function with RPB11 as ortholog
Subcomplex with rpb7, involved in STRESS response.
Shared with 1,2,3; target for transcriptional activators
Shared with 1,2,3; functions in stability and assembly
Forms subcomplex with rpb4, binds during stationary phase
shared with 1,2,3; has oligonucl and oligosach bind site
Contains zinc ribbon motif that may be involved in elongation
shared with 1,2,3
May funtion with rpb3 ortholog
shared with 1,2,3
Which subunits are essential?
rpb1 and 2
What is epitope tagging?
Insert epitope gene, rnap expresses, use antibody to bind, isolate and analyze subunits on gel.
What are componets of rnap-dna complex?
Lid and zipper/rudder/bridge/pore/mg2+
(maintain dna dissasoc./initiates dna dissaoc./lies next to active center, translocation/ntps enter, rna exits/facilitate phosphodiester formation)
Basic structure of promoter?
upstream core and coding
Upstream region has?
gene specific factors, enhancers, silencers
core region has?
assembly of polymerase, basal transcription factors
coding reigion has?
exons, introns, elongation
What are characters of class one promoters?
not well conserved, have core and upstream elements
Character of class 3 promoters?
entirely within the genes have; t1= 5srRNA/t2=tRNA/t3=non classical
Character of class 2 promoter?
Core promoter attracts gtfs and rnap2 and has TSS and proximal promoter with upstream elements.
Start and stop site on template strand?
Are there tataless promoters?
Example of initiator sequence?
py py A N t/a py py
GC and CCAAT boxes do what?
bound by TF's to stimulate transcription
How does 5' deletion series work?
Slowly degrade upstream element until reporter stops working to determine presence and character. Invivo or invitro
How does primer extension work?
invitro transcription/ hybridize oligo dt primer to mRNA and have RT go towards 5' end. Can measure transcript concentration!
How does linker scanning work?
Instead of 5' deletion, just cause mutations within the region. Identfies location of control elements, ex// is class 3 rna promoter elements.
What are enhancers?
repeats of 72bp sequence/stimulate transcription upstream of promoter/can also be internal to promoter
What is a silencer?
Act at distance, can use chromatin to condense and be inactive.
Enhancers recruit activator proteins?
Are enhancers orientation and posistion dependent?
How are enhancers and silencers tissue specific?
Rely on tissue sp. binding proteins/all cells have same genes but differ in proteins expressed/gene activity...
Order of biding of GTF's?
TF2D + TF2A>TF2B>TF2FRNAP2>TF2E>TF2H
TF2E does what?
Functions of TF2H?
Kinase and helicase
Explain nitrocellulose filter technique?
Labeled DNA in filtrate, Labeled protein in nitrocellulose, combo in paper or not.
Explain gel mobility shift
If things attach, then shifts higher in gel because heavier.
DNA footprinting to determine what?
location of binding, use dnase to slowly degrade over time, radiolabel to see where protein bound.
Class 1 factor componets?
only 2 TF with rnap/core binding: SL1 --- TBP and tafs/upstream promoter element---ubf or uaf
class 3 factors (trna) componets?
tf3b and tf3c REQUIRED, tf2a has zinc finger motifs for tight dna complex
tf2d has what subunits?
TBP: binds minor groove of tata box. (universal TF, even in tataless) 13 TAFs--taf 1 and 2 recognize other taf to combine.
TAF1 is hat, kinase?
TF2B essential for binding RNAp2?
yes, establishes TSS.
Domains of TF2B?
C= tbp bends dna and wraps around this domain
N=binds rnap2 and posistions at tss
TF2E enhcances phospho ability of H?
2 complexes on TF2H?
protein kinase (phospho CTD of RNAP2)
Helicase/ATPase (unwinds dna at tss for bubble)
helicase activity assay?
label only one, combo at top and seperate at bottom.
Preinitation complex forms with HYPOphosphorolated form of rnap2?
ctd has yspysps repeats?
rnap11a is unphospho?
rnap2o is phospho?
whic serine phospho during elongation?
which p added to which S to initate elongation?
What does TF2S do?
stimulate transcripion elongation
rnap2 can be paused and stablilized by Nelf, dsif and reversed via ptef-b?
tf2s stimulates rnase activity?
expansion of bubble will release or hold stalled poly?
releases it to clear promoter
TAT engages host rna poly?
activators can stimulate or inhibit transcription by rnap2?
the structure of an activator is made of atleast two of which categories?
dna binding, dimerization, transcription activation, hormone binding.
What are the three subcategories of dna binding domains?
zinc containing molecules (alpha helix in major groove)--zinc fingers, modules near hormone receptor, cys modules.
bZIP and BHLH---highly basic DNA binding motif linked to one/both of protein dimerization domains leucine zipper/ helix-LOOP-helix.
zinc fingers determine which dna sequence to bind precisely and interact with major groove, t/f.
Homeodomains are helix-LOOP-helix family.
the zipper in bZIP is a ---- motif
dimerization. Grasps major groove.
What are the types of transcription activation domains?
acidic/glutamine rich/proline rich
Features of GAL4 activator?
controles genes that metab. galactose/contains zinc module dna binding domain and dimerization domain/short alpha helix enters dna major groove.
Hormone binding domain features?
interact with endocrine signaling molecules/protein and endocrine molecule become activator by binding hormone response element
hormone binding domain classes?
: bind ligand in cytoplasm then migrate to nucleus.
: stay in nucleus w/ ligand activated without inactive/repressed
: orphans/not identified.
activator domains act independently? ex//?
True, gal4 with dna binding will work even if paired with a different dna binding (lexA)
What are the functions of activators?
Basal level transcription not enough, need activator to boost higher level using RECRUIT. Euk. use activators to recruit RNAP to promoters.
2 models of recruitment?
stepwise or single unit.
Ways in which activators can interact?
dimerization/transcription factories/complex enhancers/architectual transcription factors/enhancesome.
Nuclear sites where transcritpion of multiple genes occurs.
enable a gene to respond to diff. combo of activators
bending of dna to allow other activators to join is example of what?
architectual transcription factors.
nucleoprotein complex of activators bound to an enhancer.
mechanisms for activators to act at a distance?
what did catenane experiment demonstrate?
that enhancers don't have to be on same dna. can act over distance.
what is 3C, how does it work?
chromosome conformation capture: used to see how chromatin was modeled when isolated/ crosslink--digest--ligate--reverse cross--ligation products--RT to copy
properties of insulators?
protect gene from activation by nearby enhancers.
stop other chromatin invasion of target gene.
2 models of insulators?
protein on insulator interaction between strands
insulators flanking enhancer to isolate and sequester it.
insulators next to eachother to cancel and induce promoter activity.
what are coactivators?
proteins that increase expression by binding to actiavtor and stabilizing basal transcription complex.
ex// of activators?
examples of coactivator
modification examples to regulate regulators?
phosphorolate---interact with coactivators to stimulate transcription.
polyubiq---marks proteins for proteosoamal degreadion. Monoubiq---activates
sumoylation---marks P's for sequester where no activity.
methylation/acetylation/deacetylation--modulate in both directions.
therapeutic examples for HIV?
TAR rna decoys/neutralizing antibodies/attenuated viral vaccines/t cell vaccines.
ccr5 knockout mediated by?
5 componets of histones
are histone genes repeated?
How do histones affect modification?
What is a nucleosome?
core of histones around which DNA winds twice (146bp).
Which componets of histone are dimers?
H2A and B.
The histone tail is rich with ---- residues.
What are the two models of chromatin folding?
solenoid and tetranucleosome
Histones regulate activity not just structural?
How does H1 cause repression?
binding to linker dna between nucleosomes that happen to contain tss.
sp1, gal4, gaga factor act as....
what does acetylation of histones do?
removes positive charge of histones relaxing structure and increase transcription.
what does deacytelation do?
tighten grip of histones on dna to stabilize and reduce transcription.
What does HAT mean? What do they do?
Histone acetyl transferases, add acetyl groups from donor acetyl coa to CORE HISTONE TAILS.
acetylation occurs where?
cytoplasm and the nucleus.
A: in nucleus, gene regulation, looseining of nucleosome control reigions. Bromodomain attract.
B: Found in CYTOPLASM, h3 and h4 to assemble into nucleosomes. later removed.
repressors bind to dna sites and interact with co repressors which in turn bind ----
corepressors interact with unliganded RAR-RXR receptor?
results in nucleosome free zones in control regions of active genes.
Active genes are dnase sensitive why?
Have sensitive regions of relaxed chromatin.
Remodeling complexes mobilized nucleosomes but can also repress by?
changing confirmation to tighten or not.
Four classes of remodeling complex?
swi/snf: all share atpase BRG1 and BAFS
ISWI: have SANT and SLIDE domains for binding to nucleosomes
All remodeling classes do what?
alter core structure to make dna accesible to activators/repressors/nucleases.
Human IFN-beta gene activated under what?
What does IFN-beta gene recruit? and why?
swi/snf to remodel chromatin around tss.
What does histone code say?
histone tails are acetylated etc. Combo of mods in given nucleosome near control region affects the efficency of that genes transcription. The code is EPIGENETIC.
epigenetic code does not affect dna sequence?
Heterochromatin is inaccesible?
What does SIR stand for?
silencing information regulator
Order of SIR silencing example?
RAP1 binds Telomere specific site, then sir3, 4, and 2.
sir 3 and 4 interact with --- directly
H3 and H4.
acetylation of h4-k16 prevents what?
interaction with h3 and blocks heterochromatin formation.
histone methylation via?
Methyaltion of histone can have repression and activation via:::
Me to h3k9 (p1 to mt)
me to h3k4 so that h3k9 is blocked.
FACT subunits and function?
facilitiates chromatin transcription by rnap2 elngation through NUCLEOSOMES. has 2 subunits: spt16 and ssrp1 --- binds h2a/b dimers and h3/4 tetramers.
HIV infects which cells?