RR5: Proteins that regulate transcription - Activators

Question 1

what ATP dependent reaction is TFIIH responsible for?

Answer 1

phosphorylation of CTD

Question 2

what helps pol II determine the direction of transcription?

Answer 2

the TATA box binding by TBP, as part of TFIID
the TATA box makes the decision more efficient and rapid

Question 3

is TBP required for all genes, even if they do not have a TATA box?

Answer 3

yes, but if there is no TATA box you will have divergent transcription

Question 4

what binds to promoter proximal elements and what happens after binding?

Answer 4

recognised and bound to by DNA binding proteins
influence transcription reaction at/around the transcriptional start site
make the transcription process more efficient
cis acting elements

Question 5

how can EMSA be used to determine whether a specific element interacts with a protein or not?

Answer 5

depends o the concept that a DNA fragment will migrate through an electrophoretic field in a very particular way
won’t be bothered while its moving through the field under normal circumsances
if it interacts with a protein, its migration through the gel will be changed
formation of a new complex that will hinder its movement through that gel matrix

Question 6

what does EMSA stand for and what can it also be called?

Answer 6

electrophoretic mobility shift assay
can also be called gel shift or mobility shift

Question 7

how can the segments be labelled?

Answer 7

radio-labelled at the 5’ end with oligonucleotides of known sequences

Question 8

how are the segments that are protein associated identified in a gel?

Answer 8

they move away from the unbound DNA
compare with the probe to see which one it is

Question 9

how can you further test which protein is present?

Answer 9

run it over a column and carry out liquid chromatography to collect fractions that elute
can test a small volume of each fraction to determine if a protein might be present in that fraction that was there initially in the nuclear extract

Question 10

how can we evaluate whether a given factor interacts with that cis acting element and activates transcription?

Answer 10

we can generate a cDNA that corresponds to that particular protein
drive that gene under a specific promoter
transfect the expression vector of the protein you’ve identified (candidate protein)
co transfect with expression vector that has a reporter gene in it and the appropriate sequences that would respond to that given protein
put those things together by co transfecting into a cell that behaves like a factory
once cDNA is transcribed and translated into a protein, if that protein recognises the binding site (defined cis-acting element) and put in an expression vector that’s going to activate the transcription and expression of downstream reporter gene, then it will interact and activate transcription

Question 11

what should happen if those DNA binding proteins recognise the sequence?

Answer 11

there should be an increase in the reporter gene expression
if the protein does not bind the control element, there will be a baseline reporter gene activity, and it will not go above that

Question 12

what is a good way to establish control when using this method?

Answer 12

mix up that cis element, change the sequence to see if indeed that DNA binding factor actually does activate transcription through interaction with that cis acting element

Question 13

what are the two types of transcription factors?

Answer 13

transcriptional activators or transcriptional repressors

Question 14

how do those transcription factors interact with the DNA? (identified from bacteria)

Answer 14

specific domains on these DNA binding proteins are required to interact with the DNA in the major grooves
those domains are made up of helices called recognition helices
helices interact specifically with nucleobases within those major grooves
but also other amino acids within those domains contribute to stable binding –> very often positively charged basic residues that interact with the negative phosphates in the DNA backbone

Question 15

describe an example that shows the structure of those DNA binding proteins and how they work

Answer 15

GAL4 in yeast is a very important transcription factor for activating transcription of genes that are critical for using galactose as an energy source
GAL4 interacts with UAS GAl, and if there is a TAT box and a downstream gene, GAL4 will activate transcription very efficiently
GAL4 has a clear DNA binding domain and another one that is involved in transcription

Question 16

how can those different domains be identified?

Answer 16

carry out specific mutations within the protein
show that there is a specific DNA binding domain found in the N-terminal region
without it, GAL4 can no longer interact with its UAS and transcription cannot be activated
DNA binding is critical for transcription
now remove from C terminal, transcription is no longer efficient, even though GAL4 is bound to the DNA
binding alone is not sufficient
DNA binding and transcriptional activation domain are independent

Question 17

what are the different domains in a DNA binding protein?

Answer 17

DNA binding domain (structured)
transcription activation
transcription repression
chromatin remodelling
nuclear import
protein interaction

Question 18

what is a particular characteristic of these domains?

Answer 18

they can be mixed and matched, they are universal
(from humans to bacteria for example)

Question 19

what are homeodomain proteins?

Answer 19

confer positional information in organisms (ie where antennae should grow in flies)
when mutated it gives rise to changes in positional information

Question 20

what is the general structure of zinc finger DNA binding domains?

Answer 20

cysteines and histadines coordinated with a zinc ion
form a finger with the zinc
very important for interacted with the DNA

Question 21

what are the characteristics of C2H2 zinc finger domains?

Answer 21

three or more finger units
bind as monomers

Question 22

what are the characteristics of C4 zinc finger domains?

Answer 22

bind to nuclear hormones, receptors
two finger units
bind to the DNA target as homodimer or heterodimer

Question 23

what are the characteristics of C6 zinc finger domains?

Answer 23

cysteines and histadines coordinated with a zinc ion
bind as monomers (GAL4 is one of these)

Question 24

what is the structure and characteristics of leucine zipper proteins?

Answer 24

bind exclusively as homo or heterodimers with their extended basic alpha helices, which bind to the major groove of the DNA
interact with partner through hydrophobic interactions –> form a coiled coil domain
positioned all along a single face
don’t have to be leucines, can be other hydrophobic amino acids
called basic zip proteins

Question 25

what are the characteristics of helix loop helix proteins? (HLH)

Answer 25

very similar to leucine zipper proteins however instead of an extended alpha helix they are characterized by two alpha helices, connected by a short loop
through the loop, the recognition helices are positioned on the major groove

Question 26

what is cooperative binding of unrelated classes of transcription factors?

Answer 26

can interact cooperatively with other classes of DNA binding transcription domains
can occur through interaction off the DNA or on the DNA
neither domain is individually very efficient, but together can be very powerful

Question 27

what do these different transcription factor binding sites in promoters and the different combinations lead to?

Answer 27

great diversity of transcriptional responses
homo and heterodimer formation is common
even more complex when you factor in repressors

Question 28

what is Chip-seq and how does it work overall?

Answer 28

if you want to figure out exactly the sequence that the protein interacts with
use an antibody against the protein (or epitope tag that protein)
pull down the genomic sequences that are occupied by that DNA binding transcription factor and sequence them all
use computers to analyse what the sequence might look like

Question 29

what are the steps of Chip-seq?

Answer 29

crosslink macromolecules with formaldehyde
shear the DNA into small fragments
immunoprecipitate with an antibody
NG sequencing of bound DNA
DNA will correspond to genes that are bound by the transcription factor

Question 30

what is the mediator?

Answer 30

DNA binding transcription factors interact with a giant megacomplex called the mediator through specific subunits
brings everything to RNA polymerase II and general transcription factors