lecture 20--exam 5

Question 1

what are chromosomes made of? what is the complex called?
what is chromatin made of?

Answer 1

DNA, histones, non histone proteins

chromatin

8 histone proteins with DNA around it

Question 2

what are the core histones? what is wrapped around them to form the ____ of DNA packing?

Answer 2

H2A H2B H3 H4

–DNA is wrapped around them to form octamer (2 of each) , which is the first level of DNA packing, called 10nm fiber OR nucleosome fiber

Question 3

whaat the second level of DNA packing?
what are additional levels of packing referred to as?

Answer 3

nucleosome is coiled into thicker structures, referred to as 30nm fiber

higher order structures—> scaffolds where loops of 30nm fibers are anchored

Question 4

how does chromatin restrict DNA?

Answer 4

contributes to keeping genes not being used silent + generates diverse arrays of expression patterns via use of combination of large number of transcription regulators

Question 5

what are 6 levels of regulation in eukaryotic gene expression?

Answer 5

transcription inhibition

RNA processing—> alternative splicing because diff splice variants have diff functions

RNA transport–> from nucleus to cytoplasm

RNA stability–> half life of RNA varies + stability is based on sequences in the 5 + 3 UTR regions and length of poly A tail

translation efficiency–> various amounts of polypeptide based on RNA stability + efficiency

protein activation

Question 6

what are the 3 cis-acting elements that work in eukaryotes and what are their functions?

Answer 6

promotor– TATA, which binds TBP, which is a part of basal transcription factor TF2D, after pol 2 recruitment, can start

promoter proximal elements: upstream of the TATA

enchancers: distant indépendant cis acting elements
- can be upstream or downstream or a gene, far or close OR in introns
-orientation independent, work in any orientation
-in yeast, calles upstream activation sequences UAS

Question 7

what are the 3 trans acting factors (aka transcription factors) in DNA that BIND directly to DNA?

Answer 7

basal or general TF: part of RNA pol 2 that bind promoter
-regulate lots of genes and aren’t as specific

some can bind to proximal promoter elements

regulatory TF: bind enhancers away from the promoter region
+ are very specific and regulate far less genes

Question 8

what are 3 trans acting factors than do NOT bind DNA directly, but bind proteins bound to DNA?

Answer 8

mediator—large protein that links TF’s bound to enhancers + basal transcription apparatus bound to promoter

coactivators– lack DNA binding domain
-bind TF bound to enchancers

corepreossors–lack DNA binding domain
bind + have negative affect on transcription

Question 9

what are 2 basic mechanism that Tf’s use? ( can use one or both)

Answer 9

directly interact with basal transcription machinery ( RNA pol 2) OR mediator complex

open up chromatin to make it more accessible to RNA pol 2

Question 10

what is euchromatin and heterochromatin, what are their activating + inactive mods?
what happens during cell cycle + during development ?

Answer 10

euchromatin: active + loose packed
accessible tot TF’s
less methylation in major groove
active: H3 lys4Me & H4 acetylation

hetero: tighlty packed, inactive
inaccessible to TF’s
more methylation in major groove
inactivating mods: H3lys9Methylation

cell cycle: interphase the chromatin is loose, but tight during mitosis

devlepement: can differ based on conditions

Question 11

what is an enchancesome + what does it do?

Answer 11

aggregation of multiple proteins binding other proteins + binding enhancers

direct DNA bending into a loop that brings that enchancesome into contact with:
RNA pol +
tf bound at the core promoter
proximal promoter elements

Question 12

what are pioneer factors?

Answer 12

first to bind reg modules + their binding facilitates the binding of additional transaction factors

Question 13

what does the SHH gene direct + how does it generally work ?

Answer 13

production of fingers or toes + a role in brain organization

each is controlled by diff reg proteins + TF–> one combination binds enhancer in brain cells but a different combo can bind an alternative enchanter in limb cells

Question 14

what is LCR? how does it work?

Answer 14

highly specialized enchancer elements that regulate transcription of multiple genes packaged into complexes of related genes –> ex is the reg region in B globin complex

–regulation of genes contain 4 cis acting reg sequences, HS1-Hs4 that bind proteins that direct formation of small DNA loops that bridge promoters of the B globin gene

Question 15

how does GAL system work in yeast, how are they regulated?
what does Gal 4 bind to?
what does Gal4 interact with?

Answer 15

GAL1,7,10 have their own promoter, but genes are regulated by GAL4
GAL4 encodes Gal4 regulatory protein–> an activator

–UAS, which is upstream of the 4 GAL genes

-since Gal4 protein is continuously accessible, it interacts with Gal 80 (repressor), which binds it and inactives transcription

Question 16

what are the two parts of the homodimeric Gal4?
how are Gal4 and Gal80 produced?
what causes Gal4 to be activated and get rid of Gal80?

Answer 16

1)DNA binding domain targets the UAS (similar in all)
2)activation domain is a target for Gal80

constitutively produced

–when galactose is present, Gal3 binds galactose and to Gal80, which alters Gal80 and makes it release Gal4, allowing for GAL gene transcription

Question 17

what does GAL4 being activated lead to formation of?
what happens when glucose is present?

Answer 17

mediator, which is an enchansome that forms and makes a DNA loop which contact TF2D + RNA pol to allow transcription

-Mig1 is produced, which binds to a silencer by UAS, which represses UAS from directing initiation

Question 18

what are insulator sequences?
how do they work?
possible mechanism?

Answer 18

cis acting sequences located to separate enchaners from promoters

protein binding sequences that direct enhancers to interact with the promoter and block them interacting with enhancers and other promoters
-can block enchancer action and turn off a gene

allowing formation of DNA loops with their intented target and enchancer and preventing loops between promoter + enchancer that is not its target

Question 19

what are the regulated steps between CAP and GAL4?

Answer 19

CAP– DNA binding is regulated

GAL4– always bound to DNA, exposing of activation domain is regulated

Question 20

what if regulatory sequences are not bound by histones?

Answer 20

-sequences will include linker sequences between nucleosomes + sequences with specific characteristic that prevent histones from binding

Question 21

what do chromatin remodelers do?
what doe chomratin modifiers do?

Answer 21

change the distribution of the nuclesomes by being recruited to specific sites in the chromatin by trans acting factors that bind specific DNA

add or remove methyl or acetylene groups– commonly at lysines

Question 22

what are open/ covered promoters?

Answer 22

open: constitute active genes like housekeeping genes & have a NDR (nucleosome depleted region), which does not have TAT box but an A/T tract that has ES (enhancer) that attracts transcription activators

Covered: regulated + have TATA in which nucleosomes + TAF fight for binding

Question 23

what is open vs closed chromatin?
how can we tell the difference?

Answer 23

open: DNA with nucleusomes is relaxed, allowing access

closed: reg DNA is covered by nucleosomes, making it hard to get to

using DNAse1
–cuts DNA in open chromatin but is unable to do so in closed

Question 24

what are chromatin remodelers? what 3 ways do they work?

Answer 24

protein complex that carry out chromatin remodeling by moving nucleosomes by:

sliding them along chromosome or removing them from DNA—– > uncover enhancers or promoters + associated with gene activation
-SWI/SNF complex

reorganizes nucleosomes by making the nucleosome reposition to a diff DNA sequence –> usually repress
-ISWI complex

change composition of histone Octomers–> replacing histone proteins with variant ones –> gene activation usually
-SWR1 complex

Question 25

what are writers and erasers and readers ?

Answer 25

writers: add groups -are recruited to the chromatin location by seq-specific DNA binding proteins, like activators or repressors -TF erasers: remove groups -are recruited to the chromatin location by seq-specific DNA binding proteins, like activators or repressors -TF readers: recognize the modified histones -geen activity only

Question 26

where do histone modifications take place at? what are HATS and HDACS + what does acetylation do?

Answer 26

acetylene + methyl groups are added or removed from lysines in the N terminal tail of histone 3 also can be phosphorylated -K4,K9 and K27 are targets HATS: add acetyl group , recruited by activator proteins to form euchromatin HDACS: removed acetylene groups, recruited by repressor, causing heteromchromatin -acetylation neutralizes the positive charge, so it relaxes the hold on the nucleosomes

Question 27

what are HMTS + HDMTs? what does methylation usually do?

Answer 27

HMTS: writers, adds methyl group---> makes euchromatin to heterochromatin incombination with de-acetylation HDMTS: erasers, remove methylgroups---> can open chromatin when incombination with acetylation -usually on cytosine and in major groove to repress genes and silent transposons

Question 28

what is PHO5? how is it activated? what is the access of TF like in the turned off state (high phos)? what is acetylation like in the turned off state?

Answer 28

inducible gene encoding an acid phosphatase that removes P's from other proteins low phosphate levels, turned off when levels are HIGH -when repressed, TF + RNA pol 2 access to the TATA box is blocked by the -1 nucleosome -access to activator proteins to UAS element is blocked by nucleosome -2 -Pho2 & NuA4 are present upstream of the promoter at UAS element -low level of acetylation at -1 and -4, which blocks access to activator protein + TF to PHO5 sequences

Question 29

what happens to PHO5 when phos levels fall ? what starts transcription activation?

Answer 29

when phosphate levels fall, Pho4 is unphosphorylated and imported into the nucleus -Pho4 binds Pho2, which start transcription activation -acetlyion of -1 and -4 nucleosomes via NuA4 -Pho2-Pho4 displaces the -2 nucleosome, making it unavabile for Pho4 protein -SWI/SNF displaces -1, -3, and -4 -lastly GTF proteins & RNA pol2 bind promoter + aintiate transcription

Question 30

what are examples of TFs being regulated by sub cellular localization & phosphorylation?

Answer 30

sub cellular: steroid hormone receptor is inactive in cytoplasm without steroid when steroid is present, it is exported to the nucleus where is can bind DNA & work phosphorylation: Wnt signal pathway -with no Wnt, B catenin phosphorylated & degraded -When Wnt is present, it binds to a destruction complex that does not allow B cat to be phosphorylated, so it then binds to turn on transcription

Question 31

what is the difference between GTF and Reg TF?

Answer 31

GTF: recognize promoter, recruit pol2, and don't help with regulation, then bind a lot so not really specific Reg Tf: bind enhancers, are very specific so they reg transcription

Question 32

what are co repressors in prokaryotes vs eukaryotes? purpose of methylation in both?

Answer 32

co: pro: small molecules Euk: proteins methylation: pro: host defense + mismatch E: regulation n

Question 33

what feature of GAL 80 need to be lost to result in constitutive expression of the GAL genes? what feature of GAL4 would result in uninducible expression of GAL genes? what features of GAL3 would result in uninducible expression of GAL genes?

Answer 33

gal4 protein binding domain is not working dimerization is not working gal80 gene function is lost UASg domain messed up binding to mediators not working chromatin remodelers not active, so can't form loop to start gal 80 binding domain messed up galactose binding domain messed up no self dimerization