Important review for mechanisms and experiments

Question 1

Describe the experiment to identify lac mutants.

Answer 1

1. Irradiate cells to make mutants.
2. plate mutants onto full media.
3. Replica plate onto media containing lactose as only sugar/carbon source.
4. identify mutants by comparing plates and isolate colonies.
5. determine genes
6. Identified LacY and LacZ as E. coli Lac mutants

Question 2

Describe the experiment to distinguish the roles of LacY and LacZ in either cleaving the disaccharide or transporting it into the cell.

Answer 2

1. Add 14C lactose to growth media
2. Wait
3. Wash away media
4. Measure 14C levels associated with cells
5. Lactose enters both wildtype and lacZ mutants but not lacY mutant cells.
   (exposed to autoradiography)

Question 3

Describe the experiment concerning the mutational analysis of lacY.

Answer 3

1. Clone lacY into plasmid
2. pcr amplify with primers that contain single nucleotide change in the middle
3. pcr products will have mutation
4. transform lacY mutant into lacY- cells
5. Assay for:
   i - lactose binding
   ii - H+ binding
   iii - lactose uptake into cells

Question 4

Draw or define the mechanism of lac permease activity.

Answer 4

1. Lac permease open to outside of cell (R144:E126 interact)
2. H+ binding to E269 on outside
3. lactose binding in pocket near R144:E126
4. lactose binding causes displacement of H+ to E325
5. Conformational shift - now open to inside (R144:E269 interact)
6. displacement of lactose
7. displacement of lactose causes conformational change that leads to H+ release

Question 5

What occurs when neither lactose or H+ are present?

Answer 5

Transporter flips between open and closed state

Question 6

What occurs when only lactose is present?

Answer 6

Passively transported towards its electrochemial equilibrium.

Question 7

What occurs if only H+ is present?

Answer 7

H+ either binds to outside on E269 or to E325 on inside. Conformation change to inside/outside is not allowed.

Question 8

Draw the mechanism in which glucose from the gut gets transported to the blood.

Answer 8

Refer to notes honestly

Question 9

Describe the steps in K+ transport through a K+ channel.

Answer 9

1 - Negative charged amino acids near entrance of channel attract positively charged K+ (and Na+).
2 - positive charge ions accumulate in the vestibule
3 - dehydration of K+ and replacement by carbonyl groups in selectivity-filter
4 - dehydrate K+ passes through selectivity filter onto other side
Direction of K+ transport dependent on electrochemical gradient.

Question 10

Define the mechanism by which Na+ cannot pass the K+ channel.

Answer 10

1. Negative charged amino acids near entrance of channel attract positively charged sodium
   2- positively charged Na+ accumulates in vestibule
   3 - Smaller Na+ cannot interact with all 4 carbonyl groups and therefore is not dehydrated and cannot enter

Question 11

Describe nucleoplasmin and how it relates to nuclear import.

Answer 11

Pentamer made up of identical 35 kDa monomers.
Pentamer stable in vivo and in SDS-PAGE gel (somewhat)
Found at high levels in Xenopus egg nuclei.
large 150 kDa protein

Question 12

Draw out or describe the nuclear import mechanism.

Answer 12

Refer to notes

Question 13

Draw out or describe the nuclear export mechanism

Answer 13

refer to notes

Question 14

Outline the differences between nuclear import and export.

Answer 14

refer to notes

Question 15

Describe, from NEB (including how this is caused) how the Ran gradient is re-established after mitosis.

Answer 15

Refer to notes.

Question 16

Describe the Blobel expriment for identification of a signal sequence on secreted proteins.

Answer 16

refer to notes; include purification of ER and in-vitro translation

Question 17

Draw or describe the mechanism for protein translocation into the ER.

Answer 17

refer to notes, have all possibilities, i,e, soluble protein, transmembrane, post-translational, include SRP and other proteins in answer, outline differences between co-translational and post.

Question 18

Describe the mechanism of N-linked glycosylation, including the enzyme that does this, when this is done and what oligosaccharide is added.

Answer 18

Refer to notes

Question 19

Describe or draw the protein folding mechanism in the ER including how misfolded proteins are degraded

Answer 19

refer to notes

Question 20

Describe or draw the UPR.

Answer 20

refer to notes