Exam 3

Question 1

Steps for translation elongation

Answer 1

-2nd tRNA W aa is bound to eEF1 alpha
- enters A site of ribosomes
- forms of peptide bond between the aa
-c end of first AA connected to the N end of the second AA
- use peptide transferase
- slide ribosome 1 codon toward 3’ off mRNA
-tRNA w/o aa exits e site
- tRNA W/ aa chain in P site

Question 2

Steps for translation termination

Answer 2

• stop code on is in the a site
• No tRNA is complementary and anti-parallel to stop on
• eRF1 enters a site in the entire complex is released

Question 3

What function does an antibody probe have?

Answer 3

Generate primary antibody probe

Question 4

Steps for antibody probe

Answer 4

• Inject protein/antigens into another organism/species (mouse, protein, injected into a rat)
• Organisms immune system generates antibody specific to antigen (rat anti-mouse histone antibody)
  -Collect blood and purify antibody

Question 5

What is the function of immuno fluorescence?

Answer 5

Visualize the location of a protein in a fixed cell or organism

Question 6

Steps for immuno fluorescence

Answer 6

-Fix cells
-add primary antibody
- add secondary antibody and it will bind to primary antibody and secondary has a fluorescent label
-use confocal, fluorescence and excite wavelength to admit higher wavelength

Question 7

Immuno blot/western blot function

Answer 7

-Look at protein in jail, looking at the amount of translation of an mRNA into a protein.

Question 8

Steps for immunoblot or westernblot

Answer 8

• Collect all the proteins from the cell
  -Run them on SDS Page gel and do electrophoresis
• Blot to paper
• Add primary antibody to paper
• Add a secondary antibody
  -more intense the band the more protein is present

Question 9

What is the function of immuno affinity column Chromatography/ immuno precipitation?

Answer 9

Collect any singular protein

Question 10

Steps for immuno precipitation/immuno affinity chromatography

Answer 10

• Beats with primary antibody in place them into a column
  -Add cellular lysate
  -One specific protein will bind to primary antibody
• Using a mild salt wash, wash off any other proteins
  -And medium salt wash and collect ‘ protein in a tube

Question 11

Coimmunoprecipitation meaning

Answer 11

Two proteins interacting

Question 12

Where are proteins made and what is used to make them?

Answer 12

Cytoplasm; free, floating, ribosomes

Question 13

What do proteins for the plasma membrane and lysosomes use in order to form?

Answer 13

Ribosomes that will attach to the rough ER

Question 14

What is the ER membrane made of?

Answer 14

sacs called cisternae and tubules

Question 15

What separates the cytoplasm from the ER lumen

Answer 15

The ER membrane

Question 16

What are the three parts of the ER

Answer 16

Rough, smooth and transitional

Question 17

What are the properties of the rough ER?

Answer 17

Ribosomes can attach and detach and its the site of protein synthesis

Question 18

What are the properties of smooth ER?

Answer 18

No ribosomes can attach or detach, and this is the site for lipid synthesis in detoxification

Question 19

What are the properties of transitional ER?

Answer 19

For vesicles that exit ER

Question 20

What are the two types of processes for inserting proteins into the ER lumen?

Answer 20

Cotranslational translocation, and post translational translocation

Question 21

When does cotranslational translocation occur?

Answer 21

During translation

Question 22

When does post translational translocation occur?

Answer 22

After translation

Question 23

Steps for co-translational translocation

Answer 23

-start tln w eIFs, mRNA, tRNA, ribosomes in cytoplasm
- generate NTSS recognized and bound to SRP
-SRP brings ribosomes mRNA and proteins being made
-ribosomes dock on ER membrane and NTSS inserted through translocation and into ER lumen. SRP is released.
-translation continues protein made into ER lumen
-Ribosome will detach

Question 24

Steps for post translational translocation

Answer 24

-start with complete translation with a free floating ribosome
-Protein is made in chaperone protein, binds, and prevents folding
- New protein is recognized and bound by sec 62/63 in ER membrane
-Protein is pulled through Translocon using bio bip in ER lumen

Question 25

Steps for single pass w n terminal in lamin and c terminal in cytoplasm

Answer 25

• start with co-terminal trans location
• n- Terminal of the protein in lumen with NTSS clean by signal peptidase
  -Then stop transfer sequence
• Stop transfer of protein through Translocon
  -c terminal end of the protein is in the cytoplasm

Question 26

Steps for single pass c terminal in lamen and n terminal in cytoplasm

Answer 26

-Start translation of protein
-Recognition of internal signal sequence to pull c terminal end of protein into ER lumen

Question 27

How do you know if n terminal is in the lumen?

Answer 27

If there is a NTSS connected

Question 28

How do you know the c terminal end is in the lumen?

Answer 28

When there is an internal signal sequence in the ER membrane

Question 29

What enzyme cuts the NTSS

Answer 29

Peptidase

Question 30

If N terminal is in the lumen, what is in the ER membrane first?

Answer 30

Stop transfer sequence STS