Lecture 26

Question 1

True or false: proteins are strictly units of an activation OR deactivation complex that acts on DNA.

Answer 1

False

the same protein can be a part of a repressing or activating complex

Question 2

Describe the essential, and variable characteristics of a DNA transcription factor.

Answer 2

Essential:
a DNA binding module

An activation module (to turn on the gene)

Variable:
could have a dimerization module (to form a dimer with other protein subunits)

could have a regulatory module (to regulate the transcription factor)

Question 3

true or false: gene repressors inhibit transcription when repressor and activator proteins compete for the same overlapping DNA binding site. explain

Answer 3

True

the more DNA binding sites that the gene repressor can occupy, the less available DNA binding sites there are for the activator proteins to bind to and trigger transcription

Question 4

Describe the Helix-loop-helix type of Transcription factors/DNA binding proteins

Answer 4

Helix-loop-helix: is a dimer formed by 2 subunits that both feature a short alpha chain connected by a loop to a second longer alpha chain

can occur as either homodimer or heterdimer

has the 3 domains, similar to Leucine zipper transcription factors; DNA binding domain, dimerization domain, and activation domain

Question 5

Describe the steps of CHIP (Chromatin Immuno-Precipitation) technique for identifying transcription factors

Answer 5

Add gene regulatory proteins so they bind to specific DNA sequences

cross-link proteins to DNA with formaldehyde

lyse cells and break DNA into small fragments

precipitate the DNA of interest that is bound to the gene regulatory protein using antibodies against the specific gene regulatory protein

reverse the formaldehyde cross-links (to remove the protein from the DNA of interest)

amplify the precipitated DNA of interest with PCR (you can identify the sequence after PCR if you wish)

Question 6

Describe the Helix-turn-helix type of Transcription factors/DNA binding proteins

Answer 6

Helix-turn-helix: has 2 alpha helices connected by a short chain of AA’s that make a “turn” at a fixed angle

the longer helix is the “recognition helix” that fits into the major groove of the DNA when binding occurs

symmetric dimer versions of this transcription factor bind to DNA as dimers

Question 7

the promoter is where ____ ____ and ___ ______ assemble in order to conduct transcription of a gene. The regulatory sequence is where regulatory proteins ____ and control the _____ of assemble process at the promoter.

Answer 7

transcription factors

RNA Polymerase II

bind

rate

Question 8

what is the relationship between regulatory proteins and the Locus Control region of the beta globin genes?

Answer 8

regulatory proteins bind to the LCR

Question 9

Describe the Zinc finger motif type of Transcription factors/DNA binding proteins

Answer 9

Zinc finger motif: has a DNA binding motif that includes a Zn atom and usually binds in “tandem clusters”

tandem cluster binding includes multiple zinc finger domain transcription factors with multiple contact points that helps stabilize that interaction with DNA

C2H2 (Cys-His) is an example of a zinc finger domain transcription factor

Question 10

the sequence “GATA” is an example of what?

Answer 10

a recognition sequence for regulatory proteins

Question 11

describe the different motifs of DNA regulatory proteins in terms of the types of bonds they may form with a gene regulatory protein (4 of them)

Answer 11

possible hydrogen bond donors

Possible hydrogen bond acceptors

Methyl groups

Hydrogen atoms

(typical interactions between DNA motifs and gene regulatory proteins involve 10-20 interactions)

Question 12

transcription regulators bind to DNA in nucleosomes with ___ affinity and bind to naked DNA with ____ affinity

Answer 12

lower

higher

(because the surface of the nucleotide recognition sequence may be facing inward when attached to a nucleosome, which makes it less accessible to transcription regulators)

Question 13

Beta Globin gene regulation is dependent upon a region of a gene that contains ___ beta globin genes and a ____ ____ ____. explain why the last blank is important

Answer 13

5

Locus Control Region (LCR) ; it may be upstream, but it is required for transcription

Question 14

The mechanisms by which gene regulatory proteins regulate gene transcription (4 gene activator methods and 3 gene repressors methods)

Answer 14

gene activators

nucleosome remodeling
nucleosome removal
histone replacement
histone modification (such as acetylation)

Gene repressors

repressor conducts competitive DNA binding agains the activator at the binding site

both proteins bind to the DNA but the repressor binds to the activation domain of the activator protein

the repressor binds to DNA and blocks assembly of general transcription factors

Question 15

List the ways in which transcription factors are identified and give a brief description of them

Answer 15

Gel Mobility shift assay (EMSA or electrophoretic mobility shift assay): detection of sequence-specific DNA binding proteins

Affinity Chromatography: isolate DNA binding protein and purification of sequence specific binding proteins

CHIP (chromatin immuno-precipitation): allows the Identification of the sites in the genome that a KNOWN regulatory protein binds to

Question 16

Regulation by _____ is the fashion in which DNA activation/deactivation is regulated.

Answer 16

committee

Question 17

what are gene regulatory proteins? how important are they?

Answer 17

gene regulatory proteins are transcription factors that bind and activate a gene (they bind to DNA binding motifs)

they are crucial to the regulation of protein synthesis

Question 18

describe the ways (6 of them) in which repressor proteins inhibit transcription

Answer 18

Competitive DNA Binding: repressor competes with activator for the same DNA binding site

“Masking” of the activation surface: both proteins bind to the DNA but the repressor binds to the activation domain fo the activator protein

Direct interaction with the general transcription factors: the repressor binds to DNA and blocks assembly of general transcription factors

Repressor recruitment of chromatin remodeling complexes: chromatin remodeling complex returns the promoter to the “pre-transcriptional” nucleosome state

Repressor recruitment of Histone deacetylases: deacetylased promoters are much harder to remove from the histone

Repressor recruitment of Histone methyl transferase: methylated histones, via the methyl transferase, are bound to proteins that act to maintain chromatin in a transcriptionally silent form

Question 19

list the 4 ways that gene activator proteins modify DNA in terms of it’s local chromatin structure. Why do these 4 processes make DNA “more activated”?

Answer 19

Nucleosome Remodeling

Nucleosome Removal

Histone replacement

Histone modification (acetylation to be specific)(not all histone modifications cause gene activation)

all of these methods made the DNA more “accessible”

Question 20

what are gene regulatory proteins? how important are they?

Answer 20

gene regulatory proteins are transcription factors that bind and activate a gene (they bind to DNA binding motifs)

they are crucial to the regulation of protein synthesis

Question 21

Describe the Helix-loop-helix type of Transcription factors/DNA binding proteins

Answer 21

Helix-loop-helix: is a dimer formed by 2 subunits that both feature a short alpha chain connected by a loop to a second longer alpha chain

can occur as either homodimer or heterdimer

has the 3 domains, similar to Leucine zipper transcription factors; DNA binding domain, dimerization domain, and activation domain

Question 22

Describe a potential cure for sickle cell anemia that involves gene regulation. (this is all hypothetical as we are not there yet in terms of research/comprehension)

Answer 22

using gene regulation to “switch” from adult hemoglobin to fetal hemoglobin which is usually not made after birth.

Question 23

list the 4 different transcription factor (DNA binding proteins)

Answer 23

Helix-turn-helix:

Zinc finger motif:

Leucine zipper:

Helix-loop-helix:

Question 24

DNA _____ and a mediator complex allow the gene regulatory proteins to interact with the proteins that assemble at the promoter. define the mediator

Answer 24

looping

the mediator serves as an intermediary between gene regulatory proteins and RNA polymerase II

Question 25

Describe the Leucine zipper type of Transcription factors/DNA binding proteins

Answer 25

Leucine zipper: has 2 alpha helical DNA binding domains that dimerize via hydrophobic interactions between AA side chains (leucines every 7 AA’s on each alpha helix)

Dimerization domain connects the 2 alpha helices

Activation domain is where the alpha helices “cross” and it overlaps the dimer domain

The DNA binding domain is the portion of the transcription factor that actually binds to the DNA “like a clothespin”

Question 26

Hereditary Spherocytosis is an example of what type of transcription factor being mutated?

Answer 26

zinc finger transcription factor

can be found on the Klf1 (not hugely important)

Question 27

Describe the essential, and variable characteristics of a DNA transcription factor.

Answer 27

Essential:
a DNA binding module

An activation module (to turn on the gene)

Variable:
could have a dimerization module (to form a dimer with other protein subunits)

could have a regulatory module (to regulate the transcription factor)

Question 28

Describe the steps of Gel mobility shift assay technique for identifying transcription factors

Answer 28

mix radioactive DNA from a known promoter with fragmented protein extract from the sample cell

run electrophoretic gel and view the resulting bands of protein sample attached to radioactive DNA

isolate the protein to identify it

Question 29

explain what it means for transcription factors to be “modular”

Answer 29

several experiments have determined transcription factors to be modular or “each portion of the protein has a specific job”

this was determined by removing portions of the transcription factor and observing that it could no longer complete all of it’s usual activities without specific portions of the protein

Question 30

describe the ways in which gene regulatory proteins are controlled (7 ways)

Answer 30

Synthesis: make the regulatory protein (activator/repressor)

Ligand binding: causes activation of the regulatory protein

Covalent modification-phosphorylation: causes activation of the regulatory protein

Addition of a subunit: regulatory protein won’t work without a subunit

Unmasking: removal of an “inhibitor” ; could be via phosphorylation of it

Nuclear entry: once an inhibitor is removed, the regulatory protein can enter the nucleus and become active

Proteolysis: regulatory protein is associated with a membrane, and must be “cleaved” so it can be released from the membrane

“SCALPNU ; PNU at the end”

Question 31

Hereditary Spherocytosis is an example of what type of transcription factor being mutated? what is the exact mutation that occurs? also mention what the mutated protein does.

Answer 31

zinc finger transcription factor

It changes from RER to RDR

mutated KLF1 binds to the wrong strand of DNA and stays associated as a competitive inhibitor for the normal transcription of that DNA

Question 32

Describe the steps of Affinity Chromatography technique for identifying transcription factors

Answer 32

step 1 (start broad)
run the sample through a column of many different complementary DNA sequences and wash with low-salt solution to remove any protein that does not bind to DNA

wash with medium salt to elute many different DNA-binding proteins

step 2 (get more specific)
take DNA-bound protein from step 1 and run through a column containing ONLY 1 promoter recognition sequence

medium salt wash to remove all unbound proteins

high-salt wash to elute the rare protein that was bound to the single promoter recognition sequence of choice