Lecture 9 part 2

Question 1

Direction of protein synthesis?

Answer 1

amino terminal to the carboxyl terminal

Question 2

Why are there two types of methionine tRNAs?

Answer 2

One is used for the start codon only and the other for all internal methionines.

Question 3

What is added to the methionine to make it the initiator tRNA in bacteria?

Answer 3

it is formylated (added a formaline)

Question 4

What enzyme formylates the methionine in bacteria?

Answer 4

Met-tRNA-formaltransferase

Question 5

What does N-formylmethionine do in bacteria? In humans?

Answer 5

in bacteria - used as the starting amino acid.

in humans - initiates an immune response.

Question 6

What does the 30S ribosome bind to first in initiation?

Answer 6

binds to IF-1 (binds to the A site and prevents premature tRNA binding) and IF-3 (which prevents association with 50S until the right time). I

Question 7

What guides mRNA to the correct location during initiation? And where does it go?

Answer 7

16S rRNA. To the Shine-Delgarno sequence and position the AUG in the P site.

Question 8

What is the shine-delgarno sequence?

Answer 8

used to assure proper positioning of the AUG codon of the mRNA to the P site. Occurs in the 5’ untranslated region and will react by base pairing.

Question 9

Start codon for lacI (repressor) gene?

Answer 9

GUG but F-Met tRNA will still bind to it when it is in theP site.

Question 10

Where do aminoacyl-tRNAs enter in during protein synthesis?

Answer 10

The A site

Question 11

What is the chaperone for fMet-tRNA? And what does it do?

Answer 11

IF-2. And when GTP bound it will bind with fMet-tRNA and help bind to the mRNA and ribosome complex.

Question 12

When does the 50S subunit bind to the complex?

Answer 12

After IF-2 and fMet-tRNA bind. The large subunit will hydrolyze GTP on IF-2 and cause IF-1, 2, and 3 to released. Creating the 70S.

Question 13

How many initiation factors in eukaryotes?

Answer 13

At least 9.

Question 14

Initiation in eukaryotes

Answer 14

IF factors can tie the 5’ and the 3’ ends of the mRNA and then the 5’ end is scanned for the first start codon. No shine-delgarno sequence. And no polycistronic

Question 15

What are the three elongation factors in bacteria?

Answer 15

EF-Tu (thermally unstable), EF-Ts (thermally stable), and EF-G

Question 16

EF-Tu

Answer 16

acts as a chaperone and brings in the aminoacyl tRNA when GTP bound. Enters into the A site and samples the A codon (Kd). If low Kd, then will anchor the correct one down. GTP will get hydrolyzed and EF-Tu GDP bound will leave, leaving the amino acid in the A site

Question 17

EF-Ts

Answer 17

will regenerate EF-Tu with GTP. EF-Ts has high affinity for EF-Tu GDP and will cause EF-Tu to get rid of GDP. EF-Tu and EF-Ts do not have high affinity anymore and GTP will kick out EF-Ts.

Question 18

What catalyzes peptide bond formation in prokaryotes?

Answer 18

23S rRNA molecule.

Question 19

Peptide bond formation

Answer 19

deprotonation of the amine to get a nucleophile in the A site, attacks the carbonyl carbon (proximity effect) making a covalent bond. This kicks off the fMet-tRNA in the P site, attaching the amino acid to the A site residue’s amino acid

Question 20

Translocation of amino acids in ribosome

Answer 20

tRNA in A site moves to P site and tRNA in P site moves to E site, it is then released and a new tRNA comes into the A site.

Question 21

EF-G

Answer 21

required for movement of the ribosome along the mRNA molecule, using the energy of GTP hydrolysis.

Question 22

What site do eukaryotes not have?

Answer 22

an E site

Question 23

Termination

Answer 23

A termination codon recruits three release factors (RF1-3), these factors hydrolyze the terminal peptidyl-tRNA bond, release the free polypeptide and tRNA from the P site, and dissociate the 70S ribosome.

Question 24

RF2

Answer 24

self regulated by a frameshift

Question 25

Formation of each aminoacyl-tRNA requires how many high-energy phosphate groups? And where do they come from?

Answer 25

2 and they come from the adenylylation, 2 are release from ATP when AMP is added.

Question 26

How many GTPs are needed to add each amino acid?

Answer 26

4 GTP

Question 27

Puromycin

Answer 27

Inhibitor of protein synthesis. Mimics the 3' end of a tRNA molecule and will insert itself into the A site and can form a peptide-puromycin bond that causes premature dissociation.

Question 28

Tetracycline

Answer 28

blocks the A site by binding to the ribosome

Question 29

Streptomycin

Answer 29

an aminoglycoside. causes misreading of the genetic code. At [low] it doesnt read the codons correct, at [high] it blocks the binding of fMet-tRNA

Question 30

Ricin

Answer 30

catalyzes depurination of a specific adenosine in the eukaryotic 60S ribosome, leading to a rapid and irreversible inactivation

Question 31

Where residues does phosphorylation typically occur?

Answer 31

Serine, Threonine, and Tyrosine

Question 32

Where would a posttranslational modification by adding an extra carboxyl group occur?

Answer 32

On glutamic acid

Question 33

Where would a posttranslational modification by methylation occur?

Answer 33

methylation of a lysine

Question 34

Where are signal sequences typically found on the protein?

Answer 34

at the N terminus. and are generally removed once they reach its final location.

Question 35

Typically how long are signal sequences and what is their make up?

Answer 35

13 to 36 residues long and contain 10 to 15 hydrophobic residues. 1 or more positively charged near the amino end, and a short, relatively polar sequence near the cleavage site

Question 36

How does a signal sequence work?

Answer 36

a signal recognition particle binds to the sequence, stops elongation, and directs the ribosome to the ER. This is delivered to a peptide translocation complex, and elongation resumes, putting the protein into the lumen.

Question 37

SRP (signal recognition particle) in eukaryotes

Answer 37

associates with the signal sequence and the ribosome and docks it on the ER.

Question 38

What cleaves the signal sequence?

Answer 38

signal peptidase cleaves the signal sequence and then transcription resumes.

Question 39

How many residues are in the core during a glycosylation event? And where is it transferred to?

Answer 39

14 residue core oligosaccharide is synthesized in the ER and then transferred from dolichol phosphate to a protein Asn residue.

Question 40

What happens to proteins in the golgi?

Answer 40

O-linked oligosaccharides are added and additional modificiations are made to N-linked oligosaccharides. Proteins are also sorted.

Question 41

Mitochondrial proteins

Answer 41

signal sequence is recognized by cytosolic chaperone proteins, which deliver the proteins to a protein channel in the mitochondria. Translocation through the channel happens via hydrolysis of ATP and GTP. Signal sequence is then removed.

Question 42

How to get a protein in the mitochondrial membrane?

Answer 42

a "stop transfer" sequence will stop the feeding of the protein through the channel, entrapping the protein within the membrane.

Question 43

Nuclear transport sequences

Answer 43

Nuclear proteins must retain their signal sequences so that they can always go back to the nucleus after mitosis causing lysing of the nuclear membrane.