Sept 9

Question 1

What are the main components keeping the lysogen in the lysogenic cycle?

Answer 1

The Lambda and the C2 repressor

Question 2

What keeps the bacterial host in the lytic cycle?

Answer 2

The cro protein

Question 3

What does it mean when promotor L is on? What about Promotor R?

Answer 3

L means N protein expressed and R means Cro protein expressed, both for the lytic stage

Question 4

What promotor activated for lysogenic state?

Answer 4

Promotor RM, it has the cl protein

Question 5

Promotor vs operator?

Answer 5

RNA polymerase binds to promotor to initiate transcription of the gene. A repressor binds to the operator to block gene transcription

Question 6

What is the layout of the right operators in terms of the promoters?

Answer 6

From Left to Right: OR3, then 2, then 1. 3 is inside Promotor RM. OR1 is far right in Promotor R. OR2 is between the 2 promotors.

Question 7

What does Promotor RM stand for?

Answer 7

Repressor maintenance

Question 8

What is the difference in the promotors RM and R for RNA polymerase?

Answer 8

RM ( for cl gene) has weak binding for RNA polymerase, needs activator proteins to initiate transcription. R for CRO gene does not need activator protein

Question 9

Is RNA polymerase used to express prophage DNA from virus or bacteria?

Answer 9

It is a bacterial RNA polymerase

Question 10

What are the sites and structure of the C1 repressor?

Answer 10

It has N and C domain, attached by short linker. C terminus has tetramerization and dimerization sites, N domain has activating and DNA binding region.

Question 11

How does the lambda (C1) repressor work?

Answer 11

The C1 repressor dimerizes and then cooperatively binds to operators with DNA binding sites on N domains. If it binds to OR2, then it enhances binding of RNA polymerase to OR3 (for C1 gene). The dimer also physically blocks RNA polymerase from binding to Promtor R, so no CRO expression.

If it binds to OR3, RNA poly can not bind to Promotor RM (same location) and can’t express C1 repressor, therefore RNA poly can go to promotor R and CRO is ON.

If it goes to OR1, blocks promotor R so no Cro expression. RNA poly has weak binding to Promotor RM by itself so only weakly activated C1 gene.

Question 12

How many daltons on average for an AA?

Answer 12

about 110 Da

Question 13

Does the Cro protein act independently or form dimers?

Answer 13

It forms dimers as well.

Question 14

How could you study interaction of Cro or C1 with DNA?

Answer 14

Do EMSA (electrophoretic mobility shift assay). Have native gell that does not denature DNA, the DNA bound to protein would migrate slower.

Question 15

How to study only C1 binding to CR2 and not other sites?

Answer 15

Do site directed mutatgenesis so it won’t bind to other sites

Question 16

How to tell if C1 and not CRO expressed experimentally

Answer 16

Do qPCR or Northern blot, then get mRNA created from RNA poly reading gene, this will show up on gel.

Question 17

Why is it found that most of the time OR2 and 1 have the C1 dimer bound to each, and only sometimes are all the OR’s bound?

Answer 17

OR1/OR2/OR3 in terms of affinity for binding. There is cooperative binding, once binds to OR1, using the tetramerization domain OR2 as well, it increases the intrinsic affinity of OR2. The OR3 site is hard to occupy as far away from OR2 and does not have cooperative binding as needs higher Concentration of C1 to bind to OR3. Also OR2 dimer is leaning towards OR1 so not there to interact with C1 if it binds to OR3.

Question 18

What shape fo binding affinity curve would you expect for fraction bound vs [C1 protein]?

Answer 18

Sigmoidal curve as once some C1 binds to OR1, there is cooperative binding for OR2, then back to high Conc. needed for OR3 binding too.

Question 19

How does the binding of C1 to OR1 lead to a higher affinity of RNA poly to OR3 than expected?

Answer 19

There is cooperative binding so OR2 gets C1 dimer as well forming a tetramer. As OR2 now has C1 it recruits RNA poly to Promotor RM for C1 expression. This tetramer also physically blocks CRO binding to the Promotor R (for CRO expression).