8-19 Gene Expression

Question 1

Insulator Elements [2]

Answer 1

1. Barrier short-sequence that prevents migration of heterochromatin into an active and open gene
   AND
   2.stops expression of unwanted/adjacent genes

(uses Insulator Proteins)

Question 1

Proximal DNA Elements are Orientation and Distance _____[Independent/Dependent]

Answer 1

Proximal Elements on DNA near the TATA start ARE Orientation and Distance DEPENDENT! They NEED correct distance & orientation

Question 2

Regulatory Elements (6-___nucleotide in length) are both ____ and _____ independent

• Regulatory DNA regions are ____ compared to coding DNA regions
• Regulatory elements can be ___or downstream and is found in both ____ and introns

Answer 2

Regulatory Elements (6-20 nucleotide in length/short) are both ORIENTATION [can be inverted or read either from 5’ end or 3’end] and DISTANCE INDEPENDENT!

-overall Regulatory DNA Regions are ENORMOUS (50,000 bases up/down stream from start TATA Box)
_Regulatory elements can be UP or Dwnstream and is found in both exons and introns

Question 3

wht happens when tx factors come close to the start TATA Box region of DNA?

Answer 3

DNA folds and creates loops to bring regulatory elements w/their Tx factors to the start of tx where they interact with RNA Polymerase and Mediator

Question 4

What is the Mediator complex [2]

Answer 4

BIG PROTEIN Used during START of Tx containing at least 25 individual proteins associated non-covalently
-Interacts w/RNA Polymerase and Tx factors and literally mediates message from Tx factors’ to the RNA Polymerase

Question 5

Lysine of Histone tails can be ____and _____

Answer 5

Lysine of Histone tails can be ACETYLATED(by HAT)/makes Lys neutral and provides binding site
OR
METHYLATED /doesn’t destory [+] chrge (by Methylase)

-Methylation is meant for other 
regulatory proteins (CHROMODOMAINS Tx Factors)to BIND TO !

Question 6

1) Tx Factors can bind to Chromatin___ _____ in order to help…
2) [T or F] Tx factors can bind to regulatory elements even if they’re trapped in Heterochromatin?

Answer 6

Tx factors bind to ATP-dependent Chromatin Remodeling Complexes(SWI/SNF) as well to help OPEN heterochromatin for expression.

2)TRUE! Tx factors bind to regulatory elements even buried in heterochromatin–>
SWI/SNF are bound as well to remodel and open heterochromatin—>
Reader-writer Histone modification enzymes come to acetylate DNA

Question 7

Histone Chaperones

Answer 7

Remove nucleosomes from structures in the active gene units and Changes histone isoforms to more
euchromatin-friendly isoforms during Tx initiation/activation!

Question 9

Histone Arginine side chains can be _____

Answer 9

ARG side chains of histone tails can be methylated

either symmetrically or Asymmetrically

Question 10

TFIID has 2 ____domains that associate with ____ near the start of Tx initiation

Answer 10

TFIID has 2 BROMOdomains that associate with acetylation marks of histone H3 near the start of Tx–>in addition with TATA=Critical for Gene Tx initiation!

Question 10

Each Tx Factor has a \_\_\_\_domain/module+\_\_\_\_\_\_ domain/module + \_\_\_\_\_\_\_domain/module
2)Can these be separated from one another?

Answer 10

Tx Factor= Activation Domain{DYNAMIC area that can Activate AND REPRESS genes} + Dimerization Domain + DNA Binding Domain

2)YES they can be separated and shifted around!

Question 11

Each Tx Factor has a \_\_\_\_domain/module+\_\_\_\_\_\_ domain/module + \_\_\_\_\_\_\_domain/module
2)Can these be separated from one another?

Answer 11

Tx Factor= Activation Domain{DYNAMIC area that can Activate AND REPRESS genes} +
Dimerization Domain + DNA Binding Domain

2)YES they can be separated and shifted around!

Question 12

What is a requirement of DNA in order for dimer motifs of Tx factors to bind to it?

2) What does this mean for the distancing between 2 dimers within a motif?
3) Why need 2 dimers binding to 2 major grooves of DNA at one time?

Answer 12

Tx factor dimer motifs(which are symmetrical )ONLY BIND TO PALINDROME OR KINDA SYMMETRICAL pseduoPALINDROME DNA SEQ. in successive turns (34 angstroms apart) of the major groove

2) Since dimer motifs bind to successive turns of major groove the dimers are ALSO 34 angstroms apart from one another within a motif
3) 2 binding sites = you can MULTIPLY those binding strength constants together

Question 13

1) HTH(helix-turn-helix) is a motif that’s embedded in a _____ which can be alpha ____ or ___type. It’s job is to act as the _____
2) Which part of the HTH is the “recognition helix”?

3) What is the purpose of the other part of HTH
4) how are the two terminals Connected?

Answer 13

HTH is a motif protein embedded in a DOMAIN which can be alpha-helical or beta-type Domain.
HTH motifs of a domain is the DNA BINDING part of a domain

2)The C-TERMINAL helix=RECOGNITION helix=
participates in sequence-SPECIFIC major groove DNA binding

3) other part of HTH is N-terminal helix=structural support for C-term helix
4) C-terminal and N-terminal of HTH are connected by SHORT sequence of amino acids

Question 14

1) HLH (Helix-Loop-Helix) can either be____dimers or ______
2) What is HLH Dimerization generated by?

3) Examples of HLH motifs?
4) “promiscuity” ?

Answer 14

1)HLH can either be HOMOdimers or HETERODIMERS

2) Dimerization is generated by a Leucine zipper motif in HLH motif proteins–>Hydrophobic edge every other 2 turn of alpha helix
3) Ex. of HLH motif proteins=Myc, Max, Mad and MyoD

4)Leucine zipper motifs are “promiscuous” and can bind to different groups of HLH Proteins

Question 15

1) bZIP (______) AKA____ is a __dimer or ___dimer
2) What are some examples of Tx factors with bZIP motif proteins?

3)Which part of the bZIP motif actually binds to DNA?

Answer 15

1)bZIP(basic [Arg or Lys], Leucine Zipper motif protein)
“AKA Clothespin” is a Homodimer and Heterodimer to bind to DNA

2) Ex. Jun and Fos Tx factors have bZIP motif proteins inside them
3) multiple BASIC Amino Acids [+ chrged] within the bZIP motif bind to symmetric palindromic/pseduopalindromic DNA sequences

Question 16

1) Zinc-finger motif protein
2) How far apart are AA in a pair separated from each other and How far are whole pairs separated?

3)Zn Finger Tx factor Proteins have _____Zn finger domains. What does this mean for them?

Answer 16

1)Has Zn holding structure which forms 4 ligand bonds with PAIRS of either Cysteine OR imidazole group/HIS
2)THe pairs are always 2-3 AA apart from each other)
and 12-15 AA from the other opposite pair

3)Zn finger Tx factor Proteins have MULTIPLE Zn finger domains/biding sites inside them=HIGH DNA AFFINITY & HIGH DNA SPECIFICITY

Question 17

What is the benefit of having palindrome or pseduopalindrome sequences as a DNA requisite for Tx factor motifs to bind?

Answer 17

Allow Tx factor motifs to bind to TWO Successive Major groove turns of the DNA helix simultaneously=good affinity/strength

Question 19

1) DNA footprinting
2) What are the steps for this?
3) Why do you see a “footprint” in Gel Electrophoresis?

Answer 19

1)Determines where a gene regulatory Element might exist within a gene unit

2)Label radioactively the 5’ end of the DNA fragment–>mix fragment with cell lysate–>if lysate protein binds to element it will protect the element region from nuclease enzyme hydrolysis and will prevent it from being cleaved=
3)= missing fragment (nvr cleaved) will never show up when you run Gel Electrophoresis becuz it’s still bound to lysate protein, wasn’t cleaved and never got placed in gel
You’ll see a “footprint” where an element was at

Question 19

1) CHIP(Chromatin Immunoprecipitation assay) What are the steps? [5]
2) How could we use Quantitative PCR with this?

Answer 19

1)A, cross-link/covalently link DNA element to bound tx factor proteins. B.Then lyse cell and break DNA into smaller fragments (300 nucleotides long).
C.Then add antibody to bind to Tx factor we’re interested in to see if that Tx factor actually bound to specific regulatory region. When antibody binds to its Tx factor antigen it precipitates out so if Tx factor bound–>precipitate MIGHT BE [Tx factor+antibody+DNA element ] all come out.

D. Acid reverses the cross-link which releases Tx factor and leaves DNA only. Now you hopefully have a solution of DNA that’s been bound to Tx factors=DNA elements!
E. If you can Amplify the remaining DNA in solution with PCR=likely TRUE DNA elements

2)Quantitative PCR allows you to see HOW MUCH DNA element was precipitated out & much Tx factor may have been bound

Question 20

1) What is Gel-mobile shift Retardation assay

2) Is this assay intensity based?

Answer 20

1) Label radioactively the 5’ end of the DNA fragment–>mix fragment with cell lysate(s)–>if lysate protein(s) binds to element(s) it will prevent those element regions from moving very fast in gel electrophoresis=you’ll see [radioactive DNA frag+cell extract] at top of Gel Electrophoresis
2) YES! In Gel Retardation GRTR Intensity=GRTR proteins bound

Question 22

A-Describe Reporter Gene Assays [2]

B-What do you use to Assay?

Answer 22

1)Places suspected regulatory gene element [doesn’t matter where/distant independent] INSIDE a Plasmid that CONTAINS the reporter gene.
2-Place reporter gene into cell ur interested in and If reporter gene (usually insect or bacterial) is expressed after you put that cells suspected enhancement element in it then the reporter proteins made from expression can be easily assayed

[Assay using luciferase–>will emit light if reporter proteins were made due to reporter gene upregulation from suspected enhancer element].

Question 23

What is the base range for the Proximal Promoter region?

Answer 23

Proximal = -200 to +50

Question 24

A: Base Range for the CORE Promoter region?
B: What’s special about this region?

Answer 24

A: CORE Promoter region = -40 to +40
B: CORE Promoter region is where general transcription machinery binds!

Question 25

Lysine Histone Tails are DEAcetylated by ___enzyme

2-Wht does this do to the binding of DNA to that Histone?

Answer 25

Lysine Histone Tails are DEACETYLATED by HDAC enzyme!

2)DEACETYLATION= DNA will unwind and come a loose!

Question 26

ALL tx factors bind DNA as _____

Answer 26

ALL TX FACTORS BIND DNA AS DIMERS