Translation (III)

Question 1

What must happen after protein synthesis?

Answer 1

-Polypeptides must mature

Question 2

What does protein maturation include?

Answer 2

-Folding into the proper conformation
-Forming disulfide bonds
-Combining with subunits to form a complex
-Proteolytic cleavage or processing
-Derivation of specific residues
—Typically referred to as post-transitional modifications

Question 3

Why are peptide bonds of synthesized proteins cleaved after synthesis?

Answer 3

-Removal of amino acids is required for appropriate synthesis and trafficking
-Some proteins need to be cleaved in order to function (or “activated”)
-Polyproteins, proteins that are post-translationally cleaved into component proteins
-Proteins can be inactivated or destroyed by cleavage (arguable if this is “maturation”)

Question 4

What are some examples of when removal of amino acids is required for appropriate synthesis and trafficking?

Answer 4

-Cleavage of leading fMet in bacteria (majority of proteins are processed this way
-Cleavage of leading Met in eukaryotes (important for regulation of protein degradation
-Cleavage of signaling peptide after translocation into ER or other organelles targeting sequences

Question 5

What are proproteins?

Answer 5

-Synthesized as inactive precursors (proproteins) that are later activated
-Ex: calcitoning and calcitonin-gene-related peptide
-Formation of active insulin from pro-insulin by excision of internal 33 amino acids

Question 6

What are polyproteins?

Answer 6

-Proteins that are post-translationally cleaved into their component proteins through recognition of cleavage sequences
-Components are only active upon cleavage
- Ex: proteins from many viruses
-Inhibitors of HIV protease have been effective in attenuating HIV

Question 7

What are the two separate populations of ribosomes in the cytosol? How are they different?

Answer 7

-Free ribosomes and Membrance-bound ribosomes
-Free ribosomes: synthesize all other proteins released directly into the cytosol
-Membrane-bound ribosomes (rough ER): synthesize proteins into rough ER
-Both are structurally and functionally identical and dynamic
-Differ only in proteins they are making at a given time

Question 8

What proteins are captured by the ER and when and from where are they captured (broadly)?

Answer 8

-ER captures transmembrane proteins and water-soluble proteins from the cytosol as they are being translated

Question 9

How are transmembrane proteins captured by the ER/what happens to them?

Answer 9

-Partly translocated across the ER membrane, become embedded in it
-Some remain in the ER, others go to plasma membrane or membrane of another organelle

Question 10

How are water-soluble membrane proteins captured by the ER/what happens to them?

Answer 10

How do proteins get to the right place in the cell
-Peptide sequence information helps sort proteins to the right place

Question 11

What is a signal peptide used for? Where is it? What is synthesized by? What is it recognized by?

Answer 11

-Transmembrane, lysosomal, and secreted proteins are directed to the ER membrane by signal peptide
-N-terminal
-First synthesized by the ribosome
-Recognized by the Signal Recognition Particle (SRP), a ribonucleoprotein complex

Question 12

What are characteristics of signal peptides?

Answer 12

-Vary in length from 13 to 36 amino acid residues
-Have ~10 to 15 hydrophobic amino acid residues
-Have 1+ positively charged (basic) residues, usually near the amino terminus
-Have a short, polar sequence at the carboxyl terminus
-Are cleaved to remove after transport

Question 13

What is SRP

Answer 13

-Signal recognition particle
-Ribonucleoprotein complex that plays a crucial role in protein targeting during translation
-Function is to guide newly synthesized proteins to their cellular destinations, particularly the endoplasmic reticulum

Question 14

What happens when the SRP recognizes the signal peptide?

Answer 14

-Binds the signal sequence and the ribosome
-Binds GTP and halts elongation at ~70 aminoacids
-GTP-bound SRP directs the ribosome, the mRNA, and the incomplete polypeptide to GTP-bound SRP receptors in the cytosolic face of the ER
-Peptide translocation complex (translocon) interacts directly with the ribosome and accepts the nascent polypeptide

Question 15

Steps of the ER targeting pathway

Answer 15

-Protein biosynthesis begins on free ribosomes
-After the signal sequence has exited the ribosome, it is bound by the SRP, and protein synthesis stops
-SRP-ribosome complex docks with the SRP receptor in the ER membrane
-SRP and the SRP receptor simultaneously hydrolyze bound GTPs, and protein biosynthesis resumes and the SRP is free to bind another signal sequence
-Signal peptidase may remove the signal sequence as it enters the lumen of the ER
-Protein synthesis continues as the protein is synthesized directly into the ER
-Con completion of protein synthesis, the ribosome is released
-Protein tunnel in the translocon closes

Question 16

Diagram ER targeting pathway

Answer 16

SEE slide 17 for diagram of translation III

Question 17

Protein transport from ER to final destination

Answer 17

-Proteins travel from the ER to the cis side of the Golgi complex in transport vesicles
-Sorting occurs in the trans side of the Golgi complex
-Lumenal and transmembrane proteins can be trafficked in this way

Question 18

Near localization sequences (NLS)

Answer 18

-A different sequence that targets a protein to the nucleus
-Not cleaved, to allow for repeated nuclear importation following nuclear envelope breakdown
-Can be located anywhere (not just the N-term) within the protein

Question 19

How do side chain modifications affect the protein

Answer 19

-All change the biochemical properties of the modified amino acid
-Some modify protein activity (ex: phosphorylation)
-Some form recognition marks (ex: histone acetylation)
-Some alter the structural properties (such as glycosylation)

Question 20

Glycosylation

Answer 20

-More than 50% of human proteins are glycosylated
-Can be very diverse in final structure
-Often essential to protein functions
—Can impart many unique functions to protein

Question 21

How is glycosylation linked to proteins?

Answer 21

-Linked to proteins through asparagine (N-linked) or serine/threonine (O-linked)
—Either via amide nitrogen or oxygen of side chain
—N-linked have common core structure

Question 22

Diptheria

Answer 22

-Infection by the bacterium Corynebacterium diphtheriae in the mouth or throat
-Can be life-threatening
-Don’t typically worry about because of extremely low amounts of bacteria
-Disease is almost entirely mediated by protein toxin
-Vaccines against toxin are highly effective

Question 23

Where is diphthamide used?

Answer 23

-eEF2 contains a histidine with a PTM, a diphthamide
-Synthesis requires 7 enzymes
-Diphthamide occurs only in eEF2 (not even in its bacterial counterpart, EF-G)
-Diphtamide is not essential for translation in certain mutant cultured animal cells, but may be important for fidelity
—Mutations in humans lead to cancer and developmental disorders

Question 24

Diphthamide modification

Answer 24

-Diphtheria toxin catalyzes the transfer of the ADP ribose of NAD+ to the diphthamide ring of a diphthamide residue of eEF2 to inactivate it
—Called ADP-ribosylation
-Mutating diphthamide His to Asp, Lys, or Arg inactivates translation