Lecture 20 - Proteins that Regulate Transcription

Question 1

What leads to tissue or temporal specificity?

Answer 1

Enhancers

Question 2

What do enhancers do to activate a particular gene?

Answer 2

They induce a higher frequency of bursts of transcription. A more powerful enhancer results in a higher frequency of bursts.

Question 3

What do enhancers do to affect potentially a large number of genes?

Answer 3

Enhancers as well as the proteins that interact with these enhancers form a very particular area within the nucleus where a number of domains of chromosomes come together and use that same effect in order to influence their transcriptional efficiency. They are utilizing the association of many transcription factors to affect potentially a large number of genes.

Question 4

What is responsible for increasing the rate of bursting in specific zones in order to increase the frequency of transcription?

Answer 4

Enhancers

Question 5

Chloramphenicol acetyltransferase (CAT) is a type of what?

Answer 5

Reporter Gene

Question 6

What process analyzes entire DNA regions in order to identify specific sequences (through their elimination) that might be important for activating transcription?

Answer 6

Linker Scanning Mutations

Question 7

Does transcription take place in a linear manner (in terms of time)?

Answer 7

No. Transcription takes place in bursts.

Question 8

What do dsDNA probes allow in EMSA?

Answer 8

They allow the analysis of a complex mixture of potential DNA transcription factors for one or many protein complexes that may interact with that specific regulatory region, previously identified through various strategies.

Question 9

When is EMSA used?

Answer 9

It is used after linker scanning mutation.

Question 10

What does the radiolabelled dsDNA probe in EMSA bind with so that it can be identified?

Answer 10

They bind with specific transcription factor proteins so that they can be visualized.

Question 11

What is used to analyze where DNA-protein complexes might be present in a comple mixture that has been separated out using column chromatography?

Answer 11

EMSA

Question 12

What can’t EMSA/gel shifts reveal?

Answer 12

They can’t reveal the precise sequence that is bound by the protein(s).

Question 13

What are recognition helices?

Answer 13

Recognition helices are alpha-helical domains of proteins that very often will interact with the major groove in a dsDNA molecule. Through noncovalent interactions with the bases within that major groove, these recognition helices will confer specificity to the DNA binding proteins.

Question 14

What do DNA binding transcription factors often act through?

Answer 14

Recognition Helices

Question 15

What is it important to test for eukaryotic transcription factors?

Answer 15

For eukaryotic transcription factors, it is important to test whether purified transcription factors can actually interact with the DNA sequence and subsequently turn on the transcription of a given gene.

Question 16

What two constructs are needed for an assay to test DNA binding transcription factors?

Answer 16

One construct that will give rise to the production of a protein important for binding to a given element and thereby act as a transcription factor is needed. Very often, this is a transcriptional activator (as opposed to a repressor). Another construct that has a potential X binding site (cis regulatory element believed to interact with the DNA binding transcription factor produced in the first construct) is needed. This X binding site will be put upstream of a minimal promoter that will be driving the expression of a given reporter gene.

Question 17

Suppose you want to produce an assay to test DNA transcription factors. You want to test whether GAL4 will activate transcription through its UAS (in yeasts). What must you do? What will happen?

Answer 17

You must make a plasmid containing a GAL4 vector and a plasmid containing a promoter containing the GAL4 UAS upstream of a reporter gene. Both plasmids bust be co-transfected into the yeast.

The vector will be expressed and the GAL4 protein will be synthesized. If the second plasmid contains a GAL4 binding site, the protein will go into the nucleus and interact with it, thereby activating the transcription of the reporter gene.

Question 18

What is a reporter-gene construct? What is it used for?

Answer 18

A reporter-gene construct contains a UAS, TATA box (start site), and a reporter gene.

It is used as a means of assessing the ability of a transcription factor to interact with a UAS and activate a reporter gene.

Question 19

What happens if small regions of the N-terminal of GAL4 is eliminated and transfected into cells?

Answer 19

The ability to transcribe a reporter gene is drastically changed. The variant can no longer bind to a UAS because the N-terminal region is critical for DNA binding and transcriptional activation.

Question 20

What happens if small deletions in the C-terminal of GAL4 are made?

Answer 20

GAL4 will still bind to a UAS and activate transcription. However, at 755 length, GAL4 still binds to DNA but the ability to activate transcription is dramatically reduced and at 692 length or further deletions on the C-terminus, transcription is not activated at all (yet GAL4 still binds to the UAS of DNA).

Question 21

What is required for transcriptional activation activity but not necessarily sufficient enough for transcriptional activity?

Answer 21

Binding of a transcription factor (like GAL4) to the DNA is required.

Question 22

What are transcription factors referred to as modular?

Answer 22

Transcription factors are referred to as modular because they have domains which are separable.

Question 23

What are the two key domains of transcription factors?

Answer 23

DNA Binding Domain and Activation Domain (to stimulate transcription)

Question 24

What is the function of transcription factor activation domains?

Answer 24

The function of transcriptional activation domains remains a mystery today. They are disordered domains that are associated with demixing behaviours that take place in the cell.

Question 25

What can possess domains for DNA binding, transcription activation, transcription repression, chromatin remodelling, nuclear import, and protein interaction?

Answer 25

Transcription Factors

Question 26

What does the Drosophila mutation antennapedia do? Why?

Answer 26

Antennapedia leads to the growth of feet where the antennas should normally be positioned.

This occurs because there is an important transcription factor called antennapedia that is a homeobox transcription factor (which ensures that the genes which contribute to the formation of a leg are activated in the right place) that is mutated and grows feet where it shouldn’t.

Question 27

What are homeotic transformations?

Answer 27

Homeotic transformations are changes in particular genes involved in setting up the body plan.

Question 28

What type of DNA binding domains lead to homeotic transformations?

Answer 28

Homeodomain Proteins

Question 29

What does zinc interact with in zinc fingers DNA domains?

Answer 29

Cystine and Histidine Residues

Question 30

What does the “finger” structure allow zinc finger DNA domain to do?

Answer 30

It allows them to interact with double stranded DNA in the major groove.

Question 31

What are the 3 types of zinc finger DNA domains? What do the letters and numbers in the names represent?

Answer 31

C₂H₂, C₄, and C₆.

They represent the number of cystine and histidine residues. For example, C₂H₂ has 2 cystine and 2 histidine residues.

Question 32

What type of zinc finger DNA domain contains 3 or more finger units and binds to DNA as monomers?

Answer 32

C₂H₂

Question 33

What type of zinc finger DNA domain contains 2 finger units and binds to DNA as homodimers or heterodimers?

Answer 33

C₄

Question 34

What type of zinc finger DNA domain contains 6 cysteine metal ligans coordinately bound to 2 Zn²⁺ ions?

Answer 34

C₆

Question 35

Where is leucine found in leucine zipper proteins?

Answer 35

Leucine (or a different hydrophobic amino acid) is found in every seventh position in the C-terminal region of the DNA binding protein.

Question 36

What do the hydrophobic residues in leucine zipper proteins form? What are they required for?

Answer 36

The hydrophobic residues form a coiled coil domain, which is required for (homo or hetero) dimerization through hydrophobic interactions.

Question 37

What is the main difference in structure between leucine zippers and helix-loop-helix (HLH) proteins?

Answer 37

Instead of having two separate polypeptide chains, HLH proteins have an extended alpha-helix which is chracterized by two alpha-helices connected by a short loop. This loop separates the extended helix from a DNA binding recognition helix.

Question 38

What are the two helices in HLH proteins important for?

Answer 38

One helix is very important for homodimerization and heterodimerization while the other helix is critical for DNA binding (recognition and interaction with sequence).

Question 39

Where do HLH proteins contain hydrophobic amino acids?

Answer 39

HLH proteins contain hydrophobic amino acids spaced at intervals characteristic of an amphipathic alpha-helix in the C-terminal region of the DNA binding domain.

Question 40

What is chromatin immunoprecipitation used for?

Answer 40

ChIP is used to identify DNA binding sequences.

Question 41

Suppose you have identified a particular DNA binding protein/transcription factor that interacts with your cis-regulatory element of interst (through gel shift assays). What process would you perform to determine the exact sequence to which the transcription factor binds?

Answer 41

ChIP (Chromatin Immunoprecipitation)

Question 42

How is chromatin immunoprecipitation (ChIP) performed?

Answer 42

You first obtain a transcription factor tag and an antibody attached to the tag. You then perform cell lysis. After chromatin interacts with the antibody, you can precipitate the chromatin pieces which have your protein bound to it. After sequence analysis (next gen sequencing), all the DNA which was bound to the protein of interest will be identified.

Question 43

What technique is a statistical analysis of the DNA binding sites that a given transcription factor is binding to throughout the entire genome that also identifies the location of the transcription factor binding?

Answer 43

Chromatin Immunoprecipitation (ChIP)

Question 44

What do cooperative DNA binding sites do?

Answer 44

They stabilize multiple transcription factors.

Question 45

What kind of genes are regulated multiple control elements?

Answer 45

Eukaryotic Genes

Question 46

What do protein-protein interactions favour the formation of?

Answer 46

Quaternary Structures

Question 47

What leads to a diversity of transcriptional responses?

Answer 47

The diversity is caused by the combination of transcription factor binding sites in promoters.