promoters and activation of class II promoters

Question 1

what is the TATA box?

Answer 1

a promoter sequence consisting of an A-T rich octamer located 25bp upstream of the start point

Question 2

what is TBP?

Answer 2

TATA binding protein. a component which allows binding of RNA polymerase to the TATA box

Question 3

how does TBP bind TATA?

Answer 3

binds in the minor groove, forming a saddle around the DNA, bending it by 80 degrees

Question 4

how does the density of eukaryotic rRNA remain high despite decreased stability?

Answer 4

many genes are used

Question 5

what are upstream promoter elements?

Answer 5

sequences of 4,6, and 8 nucleotides which are activator sequences

Question 6

what are enhancers?

Answer 6

islands of densely packed motifs which are independent of distance and orientation

Question 7

list the proteins which assist in successful binding of RNAP II to the promoter

Answer 7

-transcriptional activators
-architectural regulators
-chromatin modification proteins
-coactivators
basal transcription factors

Question 8

what types of domain do activators have?

Answer 8

DNA binding domain which interacts with specific nucleotide sequences
activator domain which is linked by flexible yet unstructured regions that are highly charged

Question 9

how are histone acetyltransferases involved in activation?

Answer 9

recruited by activators. histone acetyltransferase acetylates lysines, which neutralises the positive charge. this weakens DNA-nucleosome interactions

Question 10

explain how the translocator protein is involved with chromatin remodelling

Answer 10

• uses ATP hydrolysis to deform and pull DNA around the nucleosome
• result is to move DNA relative to the nucleosome

Question 11

what are co-activators and how do they differ from activators?

Answer 11

the bind RNA pol II or associated proteins, stabilising its binding to the promoter
they do not bind to DNA directly

Question 12

what are cohesins?

Answer 12

architectural regulators which hold the DNA in a loop

Question 13

why are so many transcription activators needed?

Answer 13

developmentally regulated enhancers may have low affinity to ensure specificity and come in clusters for robustness

Question 14

what did experiments involving a-amanitin discover?

Answer 14

adding a-amanitin destroys the activity of RNAP II and no mRNA is produced

Question 15

what is the purpose of GTFs?

Answer 15

aid specific and efficient elongation

Question 16

what are the GTFs?

Answer 16

TBP, TFIIB, TBIIE, TBIIF, TFIIH

Question 17

how was the importance of GTFs demonstrated?

Answer 17

DNA + 32P NTPs + pol II makes inefficiently transcribed RNA, chromatin + ‘’ hardly transcribes at all
when fractions of nuclear extract are added, RNA is transcribed efficiently and accurately

Question 18

what is the role of TFIID?

Answer 18

recognises TATA, wrenches open the DNA and binds to DNA in minor groove, forming a dimer

Question 19

what is the role of TFIIH?

Answer 19

phosphorylates heptad repeats on RBP1, enabling promoter clearance and interactions with RNA processing factor

Question 20

how does a reporter assay determine the presence of a promoter sequence?

Answer 20

luciferase is an enzyme that produces light in response to a reagent when expressed, this is expressed in the reading frame of a plasmid for expression. the light produced indicates the extent of promoter content.

Question 21

how does TBP bind to the TATA box?

Answer 21

in the minor groove of the DNA, forming a saddle around the DNA, and bending it by 80 degrees.

Question 22

what is the function of a class I promoter?

Answer 22

drives expression of rRNA genes. these are transcribed in several hundred tandem repeats, and the promoter sequences are located in the gaps

Question 23

give some examples of enhancer sequences

Answer 23

SP1 (GGGCGG) CREB (TGACGTA) oestrogen receptor (AGGTCA—TGACCT) - act as binding sites for other proteins

Question 24

list the types of protein which assist in successful binding of RNA polymerase II to one of its promoters

Answer 24

• transcriptional activators
• architectural regulators
• chromatin remodelling proteins
• coactivators
• basal transcription factors

Question 25

what do the two domains of the activator do?

Answer 25

DNA binding domain - interacts with specific nucleotide sequences
activator domain - linked by flexible but unstructured regions, highly charged

Question 26

what is the advantage of a non-structured region in activators?

Answer 26

lack of structure allows them to wrap around surfaces of proteins without high affinity

Question 27

give an example of a DNA binding domain of an activator

Answer 27

zinc finger - interacts with the double helix to make contacts with specific bases

Question 28

what is unusual about nuclear receptors?

Answer 28

class of activator which contain zinc fingers acting as a dimer. these are activated by ligands, which makes them unusual.

Question 29

what other proteins do bound activators recruit?

Answer 29

acetyltransferases
chromatin remodelling factors
coactivators

Question 30

what do acetyltransferases do?

Answer 30

histone acetyltransferases acetylate lysine, removing the positive charge and weakening DNA-nucleosome interactions

Question 31

how is the translocator a chromatin remodelling factor?

Answer 31

uses ATP hydrolysis to deform and pull DNA around the nucleosome, this requires ATP, as this process involves the loss of electrostatic interactions