Protein Synthesis

Question 1

How many possible combinations of the 4 mRNA nucleotide cases are there for each codon?

Answer 1

64 possible combinations (=4³)

Question 2

What does degeneracy mean in terms of translation?

Answer 2

The code for protein synthesis is less specialized, or degenerate

Although there are 64 possible combinations for amino acids, there are only 20 coded for

Redundancy

Question 3

Describe the wobble position and how it contributes to degeneracy.

Answer 3

The wobble position is the 3rd base in the codon sequence. In many cases, the amino acid coded for is only determined by the first two bases, and the third base does not matter

This is the source for the degeneracy of the genetic code.

Question 4

What nucleotide bases determine the 3 stop codons?

Answer 4

UAA, UGA, UAG

“U Are Annoying, U Go Away, U Are Gone”

Question 5

What are the three major properties of the genetic code?

Answer 5

1) The genetic code is universal (all organisms use same code)
2) The genetic code is degenerate
3) Some codons have additional functions (methionine to start, and stop codons)

Question 6

With the triplet codon structure of mRNA, how many different reading frames exist?

Answer 6

There are 3 different possible reading frames, but only one of them will produce a functional protein

Frameshift mutations usually cause premature stop codons that terminate protein synthesis

Question 7

What are the main steps of translation?

Answer 7

1. Charging the tRNA
2. Initiation
3. Elongation
4. Termination

Question 8

Describe the secondary and tertiary structure of tRNA molecules

Answer 8

Secondary Structure: cloverleaf structure with the tRNA looping into 3 “leaves. The middle leaf has the anticodon and the “stem” has the amino acid binding site. Many of the bases are modified.

Tertiary structure: L structure is the folded form with the anticodon on one end of the L, the amino acid binding site on the other end and the T/D loops at the “elbow”

Question 9

True or false: The anticodon is a 3 base pair sequence found on tRNA strands

Answer 9

True.

Anticodons are found on tRNA and are complimentary to the codons of the mRNA

Question 10

Which position number is the wobble position on the codon? What about on the anticodon?

Answer 10

On the codon, the wobble position is number 3

On the anticodon, the wobble position is number 1

*Always read 5’–>3’

Question 11

What enzyme charges the tRNA?

Answer 11

Aminoacyl-tRNA synthetases

Question 12

Describe how amino acids are attached to tRNA molecules

Answer 12

Catalyzed by aminoacyl-tRNA synthetases

With energy input from ATP, amino acids are adenylated by linking the carboxyl of the amino acid to a adenine.

The aminoacyl-tRNA is then formed by producing a high energy ester linkage between the tRNA and the amino acid

Question 13

How do aminoacyl-tRNA synthetases differ between eukaryotes and prokaryotes?

Answer 13

In eukaryotes, each amino acid has a unique aminoacyl-tRNA synthetase

In prokaryotes, one synthetase couples more than one amino acid

Question 14

How do aminoacyl-tRNA synthetases ensure that the correct tRNA molecule is bound?

Answer 14

The anticodon regions of the tRNA are inserted into a pocket and the shape and charge of the nucleotides in the anticodon are checked.

This is not always useful because some amino acids are coded for by 6 different codons. The 3’ amino acid accepting arm is also matched to the enzyme to ensure that the correct tRNA is bound.

Question 15

How does aminoacyl tRNA synthetase select the correct amino acid?

Answer 15

Two separate sites on the enzyme are used to ensure that the correct amino acid is attached to the tRNA.

First, the size of the amino acid determines whether or not it can fit into the synthesis site. If it is too big, then it will not fit into this site and will not be attached to the tRNA.

If the amino acid is too small, it will fit in the amino acid, but it will also fit into the editing site. Amino acids are removed from the tRNA in the editing site.

Question 16

What is the accuracy of tRNA charging?

Answer 16

1 error is made in every 40,000 couplings

Question 17

What is a Shine-Dalgarno site?

Answer 17

Shine-Dalgarno sites consist of 3-9 bases in the mRNA that base pair with the 3’ end of the small ribosomal subunit. This is located about 5 base pairs upstream of the initiator codon.

Question 18

Describe the initiation of translation in prokaryotes.

Answer 18

Initiation occurs at AUG codons with properly spaced Shine-Dalgarno sequences

The 16S rRNA base pairs with the SD sequence and positions the start codon within the ribosome.

IF2 has a bound GTP that binds to the initiating formyl-methionine tRNA

The large ribosomal subunit then attaches and the IF2-GTP is hydrolyzed in order to catalyze the bond formation

Question 19

True or false: Eukaryotic mRNA is polycistronic.

Answer 19

False.

Prokaryotic mRNA is polycystronic because ribosomes assemble directly onto each start codon. Eukaryotic mRNA is monocystronic.

Question 20

Describe the initiation of protein synthesis in eukaryotes.

Answer 20

Initiation tRNA with bound eIF2-GTP is loaded onto the small ribosomal subunit

eIF-4E and 4G bind to the 5’ cap and the polyA tail to make sure the mRNA is intact

The small ribosomal subunit then scans for the first AUG (start codon)

When the AUG is found, eIF2-GTP is hydrolyzed and the large ribosomal subunit binds to the small subunit.

The initiation tRNA is in the P-site of the ribosome at this point and protein synthesis is ready to begin.

Question 21

How is eIF2-GDP recycled?

Answer 21

eIF2-GDP is produced when eIF2-GTP is hydrolyzed during initiation

It is recycled by eIF2B, a guanine nucleotide exchange factor using a molecule of GTP

Question 22

What factor binds to the 5’ end of mRNA during the initiation of translation?

Answer 22

eIF4 (composed of eIF4E,G and A)

This confirms that mRNA is properly processed and promotes the binding of the 40S subunit to the mRNA

Question 23

Describe the mechanism for how the 40S ribosomal unit binds to the mRNA in translation initiataion.

Answer 23

eIF2-GTP binds to the met-tRNA which then binds to the 40S rRNA

eIF3 binds to the small ribosomal subunit and interacts with eIF4G which is already bound to the 5’ end of the mRNA

The small subunit is then attached to the 5’ end of the mRNA

Question 24

How does the 40S ribosome detach from eIF4 to begin scanning?

Answer 24

The hydrolysis of ATP is used to remove the eIF4 (E,A, G and B) from the mRNA

The ribsome is then free to scan along the mRNA for the first AUG sequence

Question 25

Describe how the 80S ribosome is assembled on the mRNA

Answer 25

When the ribsome reaches an AUG sequence, the anticodon of the Met-tRNA is complementary to the AUG

The eIF2-GTP is hydrolyzed causing a conformational change and the release of several initiation factors

The 60S subunit can bind to the mRNA and the Met-tRNA is found in the P-site of the ribosome

Question 26

What are the 4 binding sites of the ribosome?

Answer 26

mRNA site, A-site, P-site, E-site

Question 27

Describe the movement of tRNA’s through the ribosomal binding sites

Answer 27

tRNA’s go APE

A–>P–>E

Question 28

How do tRNAs get from one site to the next in the ribosome?

Answer 28

Conformational changes of the ribosome move the mRNA and tRNA 3 nucleotides

After the shift, the A site is open for the incoming aminoacyl-tRNA to bind to its complimentary codon and form a peptide bond with the amino acid located in the p-site

Question 29

What are the two elongation factors that participate in protein synthesis? How do they work?

Answer 29

EF-Tu and EF-G

EF-Tu-GTP binds to a charged tRNA as it enters the ribosome. The GTP is hydrolyzed and EF-Tu exits the ribosome.

EF-G-GTP binds near the A site and accelerates the movement of tRNAs to the P and E sites. Hydrolysis of this GTP catalyzes the confrmational change needed to move the tRNA and advance the mRNA by one codon.

Question 30

During elongation, what mechanism is responsible for the near-perfect accuracy of ribosomal translation?

Answer 30

Two lags (GTP hydrolysis and EF-Tu exiting) provide an opportunity for incorrectly bound tRNA to exit the ribosome prior to incorporation into the polypeptide chain.

Question 31

Describe how translation is terminated.

Answer 31

A stop codon (UAA, UGA, UAG) is reached

A release factor binds to the ribosome and forces the peptidyl transferase of the ribosome to catalyze the addition of a water molecule to the peptide chain instead of an amino acid

The carboxyl terminus from the tRNA is now free and the completed protein is released

Question 32

What steps in protein synthesis contribute to its fidelity?

Answer 32

1) Aminoacyl tRNA synthetase must recognize the correct tRNA
2) Aminoacyl tRNA synthetase must select the correct amino acid
3) mRNA must be fully processed prior to initiation
4) Ribosome matches mRNA codon to tRNA anticodon, stronger interactions exist in correct pairings
5) GTP hydrolysis and release of EF-Tu provide 2 lags allowing time for incorrect tRNA to be released

Question 33

What are the differences between transcription and translation in prokaryotes versus eukaryotes?

Answer 33

PROKARYOTES:

• transcription and translation occur in the same compartment and occur simultaneously
• no cap/tail is on mRNA
• only 1 type of RNA polymerase
• polycystronic translaton
• 70S ribosome composed of 3 subunits

EUKARYOTES:

• transcription occurs in the nucleus, translation in the cytoplasm
• mRNA is capped, spliced, and polyadenylated before entering the cytoplasm
• 3 different RNA polymerases produce 3 different RNA types
• monocystronic translation
• 80S ribosome composed of 4 subunits

Question 34

How does puromycin work?

Answer 34

Molecular mimicry

Puromycin has a structure very simlar to tRNA

During protein synthesis, puromycin can bind to the A-side of the ribosome and a peptide bond is formed between puromycin and the peptide chain.

The protein synthesis is then stopped because puromycin lacks the high energy ester linkage to add additional amino acids

Question 35

How does hsp70 work?

Answer 35

hsp 70 is a molecular chaperone that helps proteins fold

hsp70 molecules interact with exposed hydrophobic patches on proteins immediately after they leave the ribosome

ATP hydrolysis causes hsp70 to bind tightly to the protein.

Repeated binding and release helps proteins fold.

Question 36

How does hsp80 work?

Answer 36

hsp80 is a barrel shaped protein complex that helps proteins fold properly

hydrophobic amino acids interact with the edge of the barrel

The protein enters the barrel into an environment that encourages folding

If the protein is not properly folded, it will be released and degraded in a lysosome