Topic 2 (DNA-protein interactions)

Question 1

What three components make up a nucleotide?

Answer 1

Ribose sugar, nitrogenous base, and at least one phosphate

Question 2

Where does the glycosidic bond attach between the ribose sugar and base?

Answer 2

C1 of the sugar and N9 of purines or N1 of pyrimidines

Question 3

What are the characteristics of a purine?

Answer 3

Double ring, aromatic, 5 nitrogens

Question 4

What are the characteristics of a pyrimidine?

Answer 4

Single ring, aromatic, 2-3 nitrogens

Question 5

What are the characteristics of the DNA double helix backbone?

Answer 5

Sugar-phosphate, negatively charged, polar, hydrophilic

Question 6

Where are the nitrogenous bases found within a DNA double helix?

Answer 6

Inside, stacked on top of each other

Question 7

What is the width of the minor groove?

Answer 7

12Å (1.2nm)

Question 8

What is the width of the major groove?

Answer 8

22Å (2.2nm)

Question 9

What is the angle associated with the major groove?

Answer 9

210°

Question 10

What is the angle associated with the minor groove?

Answer 10

150°

Question 11

From where are the angles measured for the major and minor grooves?

Answer 11

Between the glycosidic bonds of a base pair

Question 12

How can you determine the base pairing when looking at the major groove?

Answer 12

Look at the order of nitrogens and oxygens exposed to the major groove. If blue, red, blue, it’s GC. If blue, blue, red, it’s AT. If red, blue, blue, it’s TA.

Question 13

True/False? The operator sites for lambda are palindromic

Answer 13

True

Question 14

How many peptide bonds can an amino acid form?

Answer 14

Two. One on each terminal

Question 15

How is the structure of protein different from DNA?

Answer 15

The backbone is found inside rather than outside and the R groups protrude outwards

Question 16

What are the two common secondary structures found in proteins?

Answer 16

Alpha-helices and beta-sheets

Question 17

How many helices make up the N domain of the lambda repressor? Which ones are of interest to us?

Answer 17

5. 3 and 2 are most important for our purposes

Question 18

What motif is used to help the lambda repressor bind to DNA?

Answer 18

Helix-turn-helix

Question 19

Which helix in the N domain of lambda repressor interacts with the major groove?

Answer 19

3

Question 20

Which helix in the N domain of lambda repressor helps to position the protein onto the DNA?

Answer 20

2

Question 21

What is the function of helix 3 in lambda repressor?

Answer 21

Recognizes and fits into the major groove

Question 22

What is the function of helix 2 in lambda repressor?

Answer 22

Helps to position helix 3 into the major groove

Question 23

How does the repressor further enhance DNA binding after helix 2 and 3 are in position?

Answer 23

The repressor arms wrap around the operator

Question 24

True/False? Cro has the same secondary structure as the N domain of the lambda repressor

Answer 24

False. It has 3 alpha-helices and 3 beta-sheets

Question 25

Cro uses helices 2 and 3 for the same function as the lambda repressor. What does this imply about their sequences?

Answer 25

The sequence that makes up their primary structures are conserved

Question 26

Which amino acids are conserved between helix 3 of both the lambda repressor and Cro proteins?

Answer 26

Glutamine (Gln) and Serine (Ser)

Question 27

How do Gln and Ser stabilize their proteins when interacting with DNA?

Answer 27

They are polar and can H-bond with the DNA backbone

Question 28

How does the Ala residue in helix 3 of the lambda repressor create a stable protein-DNA interaction?

Answer 28

It creates a nonpolar (hydrophobic environment) and interacts with the hydrophobic inside of the DNA stand

Question 29

How does the Lys residue in helix 3 of the Cro protein create a stable protein-DNA interaction?

Answer 29

It has a positive charge and a long chain to interact with the negatively charged sugar-phosphate backbone of the DNA

Question 30

Which nucleotide does Gln of helix 3 interact with? How?

Answer 30

Adenine. It has two hydrogen-bonding interacts with this base

Question 31

What role does the Gln in helix 2 play in both the repressor and Cro?

Answer 31

It interacts with the DNA backbone with its partial positive charge

Question 32

What role does the Ala in helix 2 play in both the repressor and Cro?

Answer 32

It shares hydrophobic interactions with helix 3’s Val and Ile, respectively. This allows the protein to maintain its shape

Question 33

What is the sigma factor? What is its role?

Answer 33

The sigma factor interacts with the promoter region to recruit RNA polymerase

Question 34

What is the Pribnow box?

Answer 34

The -10 sequence, included in RNA pol binding site

Question 35

Why is RNA pol exclusively bound to either PRM or PR?

Answer 35

PRM requires a repressor to be bound in the OR2 site, which blocks RNA pol from binding to PR. PR is always on otherwise

Question 36

Why is PR blocked and PRM active when a repressor is bound to OR2?

Answer 36

PR has a 3 nucleotide overlap with OR2, while PRM has a 2 nucleotide overlap. This single nucleotide difference prevents RNA pol from binding PR but enhances binding at PRM

Question 37

Where are the 3 residues that positively promote transcription initiation found on the lambda repressor?

Answer 37

Two on helix 2 and one on the link between helices 2 and 3