S1B4 - Protein Synthesis

Question 1

What are the five stages of protein synthesis?

Answer 1

There are five stages of protein synthesis.

1. Activation
2. Initiation
3. Elongation
4. Termination
5. Posttranslational modifications

Question 2

In what direction is mRNA translated from?

Answer 2

The messenger RNA is translated from the 5’ end to the 3’ end.

Question 3

During elongation of a polypeptide, what is the energy source for transporting a tRNA-aminoacyl complex into the ribosomal A site?

A) Guanosine triphosphate

B) tRNA-amino acid bond

C) Adenosine triphosphate

D) ELectrochemical gradient

E) Glycolysis

Answer 3

Guanosine triphosphate

Two steps during elongation require energy: “loading” of tRNA into the ribosomal A site and translocation of the newly elongated polypeptide from the A site to the P site. These steps each consume 1 GTP (not ATP).

Question 4

What is required for binding of a new aminoacyl tRNA to the ribosomal A-site during protein translation?

Answer 4

Binding of the aminoacyl tRNA to the A-site requires GTP hydrolysis (think “G” for Gripping, i.e. binding) and elongation factors (EFs).

Question 5

Which is the eukaryotic ribosome’s large subunit?

Answer 5

Prokaryotic large subunit: 50S. Eukaryotic large subunit: 60S.

Mnemonic:”prOkaryotic=Odd=30S (small), 50S (large), 70S (whole ribosome complex), Eukaryotic=Even=40S (small), 60S (large), 80S (whole ribosome complex)”

Question 6

Except for the initiator tRNA, to which ribosomal site do all tRNAs bind?

Answer 6

Initiator tRNA is the only tRNA that can bind to the P-site; all others bind to A-site.

Question 7

How many GTP molecules are consumed per elongation cycle in the process of translation?

Answer 7

As peptidyl transferase catalyzes the new peptide bond, the ribosome translocates one codon downstream, moving the latest tRNA and the newly elongated polypeptide chain into the P-site and emptying the A-site.

• Translocation consumes 1 GTP (think “G” for Going places, i.e. translocation). So, two GTP are used per cycle, one for binding (“Gripping”) of the aminoacyl tRNA and one for translocation (“Going places”) of the ribosome along the mRNA.

Question 8

How does translational termination occur?

Answer 8

Termination: Stop codons are recognized by protein release factors, which release the polypeptide from the ribosome and cause the ribosomal subunits to dissociate.

Question 9

In protein translation, how many high-energy phosphate bonds are used per amino acid?

Answer 9

In all, translation uses 4 high-energy phosphate bonds per amino acid:

• 2 during aminoacylation (tRNA charging): ATP → AMP (A for Activation)
• 1 during tRNA “loading” into the A-site: GTP → GDP (G for Gripping)
• 1 during translocation: GTP → GDP (G for Going places)

Question 10

During initiation of translation in eukaryotes, which ribosomal subunit binds upstream of the 5’ end of the gene?

A) 40S subunit

B) 30S subunit

C) 23S subunit

D) 60S subunit

E) 50S subunit

Answer 10

40S subunit

Eukaryotes –> Even subunit

Small subunit binds first.

Question 11

Which ribosomal site does the initial Met tRNA enter during translation?

Answer 11

The small ribosomal subunit is joint by the large subunit, initiation factors, and the initiator tRNA. The initiator tRNA (Met or fMet) enters the P-site. The initiation factors dissociate from the ribosome-mRNA complex once initiation is complete.

Question 12

Termination of eukaryotic translation requires which of the following?

A) Stop codon recognition by ribosomal E-site

B) Pairing of stop codon with tRNA-threonine

C) Pairing of stop codon with tRNA-methionine

D) Protein release factors binding to stop codon

E) Stop codon recognition by ribosomal A-site

Answer 12

D - Protein release factors binding to stop codon

Protein release factors bind to stop codons; when the ribosomes reach these factors the subunits dissociate and the polypeptide is released.

Question 13

In prokaryotes, which event occurs when ribosomes reach a start codon?

A) N-Formylmethionine-tRNA enters the ribosomal A-site

B) S-adenosyl methionine-tRNA enters the ribosomal A-site

C) Methionine-tRNA enters the ribosomal P-site

D) N-Formylmethionine-tRNA enters the ribosomal P-site

E) S-adenosyl methionine-tRNA enters the ribosomal A-site

Answer 13

D - N-Formylmethionine-tRNA enters the ribosomal P-site

The AUG codon (start codon) encodes for methionine. In eukaryotes, methionine is used. In prokaryotes, fMet (N-formylmethionine) is used. This first tRNA enters the ribosome at the P-site.

S-adenosyl methionine is a methyl donator molecule.

Question 14

Which is the prokaryotic ribosome’s small subunit?

Answer 14

• Prokaryotic small subunit: 30S. Eukaryotic small subunit: 40S.
• Mnemonic:”prOkaryotic=Odd=30S (small), 50S (large), 70S (whole ribosome complex), Eukaryotic=Even=40S (small), 60S (large), 80S (whole ribosome complex)”

Question 15

Humans transcribe 45 unique tRNAs, but utilize 64 codons in their genetic code. What accounts for this difference?

A) The tRNA cloverleaf structure confers additional codon specificity

B) The codon’s 3rd nucleodide is allowed to mismatch with the tRNA 3’ CCA sequence

C) The codon’s 2nd nucleotide is allowed to mismatch with the tRNA 3’ CCA sequence

D) The codon’s 3rd nucleotide is allowed to mismatch with the tRNA anticodon

E) The tertiary structure confers additional codon specificity

Answer 15

D) The codon’s 3rd nucleotide is allowed to mismatch with the tRNA anticodon

The “Wobble hypothesis” accounts for the differing number of tRNAs and codons. In essence, the codon’s 3rd position is allowed to “wobble”, meaning that the tRNA anticodon-to-codon pairing is allowed to mismatch at this position.

Question 16

What is the “wobble” hypothesis of mRNA/tRNA pairing?

Answer 16

Wobble hypothesis: the 3rd position of the mRNA codon isn’t as critical to pairing and is allowed some “wobble” with respect to nucleotide base pairing with the tRNA. tRNAs that code for the same amino acid often differ in this “wobble” position.

Question 17

What enzyme is responsible for ensuring tRNAs are matched to the correct amino acid? When does this proofreading occur?

Answer 17

Aminoacyl-tRNA synthetases proofread both before and after charging a tRNA with an amino acid. If the wrong amino acid is on the tRNA, the covalent bond is hydrolyzed.

Question 18

What molecule provides the energy for tRNA aminoacylation (“charging”)?

Answer 18

Aminoacylation (aka “charging”) uses ATP. It gives a phosphate group (hence “charged”) that later provides energy for peptide bond formation; aminoacylation converts ATP → AMP (2 phosphate bonds).

Question 19

What is the cellular consequence of a mischarged tRNA?

Answer 19

In the event that an error in proofreading occurs, a mischarged tRNA is formed. Mischarged tRNAs insert the incorrect amino acid into a growing polypeptide chain because they retain the ability to read the codon corresponding to the amino acid they should have been matched to.

Question 20

What trinucleotide sequence on tRNA forms a peptide bond with the amino acid to be matched with it?

Answer 20

The 3’ end of tRNA has a CCA sequence that accepts the amino acid to be matched with it.

In the 3D image, the CCA is shown in orange. Mnemonic: CCA “Can Carry Amino Acids”

Question 21

What are the shapes of the secondary and tertiary structures of tRNA?

Answer 21

The secondary structure is “cloverleaf,”while the tertiary structure is “L” shaped.

The “bottom” of the cloverleaf houses the anti-codon, which pairs with mRNA codons when brought together in a ribosome.

Question 22

How many different amino acids does each aminoacyl-tRNA synthetase recognize?

Answer 22

There is one aminoacyl-tRNA synthetase per amino acid. Because the genetic code is degenerate, some amino acids use multiple different tRNAs that recognize different codons that code for the same amino acid. Thus, each aminoacyl-tRNA synthetase may recognize multiple tRNAs but only one amino acid.

Question 23

What region of tRNA binds to the ribosome?

Answer 23

The T Ŷ C loop- this is where the tRNA binds to the ribosome.

Question 24

What does the D loop on tRNA contain and what is it recognized by?

Answer 24

The D loop – this loop contains a high percentage of dihydrouridine residues (this is recognized by the enzyme tRNA synthetase).

Question 25

On which end of a tRNA molecule do aminoacids bind?

Answer 25

The 3’ end of the molecule terminates with the sequences C-C-A. This is the amino acid binding site.

Question 26

What is inosine?

Answer 26

Wobble hypothesis – F. Crick (1965). The third nucleotide of the codon is degenerate (helps explain degeneracy of the code). Sometimes unusual base pairing takes place between the third base of the codon and the first base of the anticodon.

U in anticodon recognizes A or G in codon.

G in anticodon recognizes C or U in codon.

I (inosine) in anticodon recognizes U, C, or A in codon.

The inosine nucleotide is formed by adenosine deaminase after tRNA is formed on certain tRNAs.

Question 27

What does activation of tRNA involve? What group of enzymes catalyze this?

Answer 27

Activation involves the coupling of an amino acid to its corresponding tRNA. These reactions are catalyzed by a group of enzymes termed the amino acyl tRNA synthetases.

Question 28

What type of bond is formed between a tRNA and an amino acid after activation? Where is this bond?

Answer 28

The ester bond between the amino acid and the tRNA molecules is a high energy bond. The amino acid is bound to the 2’ or 3’-OH group of the ribose of the terminal adenylate residue of tRNA.

Question 30

How many tRNA synthetases are there in humans?

Answer 30

Number of tRNA synthetases is 20. (one for each amino acid) A single synthetase is able to recognize all the tRNA molecules that can be linked to the same amino acid.

Question 31

If an amino acid that is bound to a tRNA gets chemically altered, will it be incorporated into a protein?

Answer 31

Role of the amino acid in recognizing the anticodon for that amino acid: None. If an amino acid attached to the tRNA is chemically altered, the altered form will be incorporated into the protein.

Question 32

What three initiation factors are involved for prokaryotic initiation?

Answer 32

Three initiation factors are involved for prokaryotic initiation: IF 1,2,3

1. IF1 and 3 bind to the free 30S subunit .
2. mRNA then also binds to the 30S ribosome subunit.
3. IF2 (GTP) binds to tRNAf^met which binds AUG at the P site.
4. IF2 is lost as GTP is hydrolyzed and 70S ribosome is formed and IF1 and 3 are lost.

Question 33

What do you need to know about eukaryotic protein syntesis initiation?

Answer 33

Eukaryotic protein synthesis initiation has more and different factors. Know that eIF-4E (eukaryotic initiation factor 4E) is an mRNA cap binding factor which is essential for lining up the start codon on the ribosome.

Question 34

What are the A, P & E sites in a ribozome complex?

Answer 34

A&P & (E sites): The reassembled ribosome possesses two sites at which tRNA with their attached amino acids hybridize through their anticodons to the mRNA codons: P is the peptide site, A is the acceptor site, while E is an exit site for empty tRNA ejection.

Question 35

What is the Shine-Dalgarno sequence?

Answer 35

Recognition: Prokaryotes: Between the 5’ end of the mRNA and the initiation codon (AUG) is a sequence of nucleotides called the leader sequence. Within this sequence is the Shine-Dalgarno sequence (AGGAG, consensus) which is located approximately 10 nucleotides 5’ to the initiation codon AUG. Thus, the SD sequence permits binding and proper orientation of mRNA with ribosome. This orientation specifically locates the AUG codon for initiation and distinguishes it from other AUG codons in the translated regions.

Question 36

Why is eukaryotic mRNA monocistronic?

Answer 36

In eukaryotes the AUG nearest the 5’ cap structure is the initiating codon. Since there will be only one of these per mRNA molecule (i.e., only one 5’ end), only one protein can be made per eukaryotic mRNA molecule; i.e., eukaryotic mRNA is monocistronic.

Question 37

What determines the relative efficiency of eukaryotic mRNA translation?

Answer 37

The relative efficiency of eukaryotic message translation is determined by Kozac Consensus Sequence surrounding AUG start site. Those messages which are close in sequence to Kozac sequence, GCCGCCA/GCCAUGG will translate efficiently.

Question 38

What are the three stages of elongation during protein synthesis?

Answer 38

There are three stages of elongation which operate in a cycle.

1. Binding – the correct amino acyl-tRNA is positioned in the ribosomal complex.
2. Peptide bond formation.
3. Translocation – the ribosome is moved downstream relative to the mRNA by one codon; i.e., moved toward the 3’-end of the mRNA.

Question 39

If you see EF-Tu, whoat should you think about?

Answer 39

Think about the binding stage of elongation in protein synthesis.

The aminoacyl tRNA possessing the anticodon to the codon in the A site forms a complex with the elongation factor EF-Tu and GTP. This complex then binds to the ribosome such that the aminoacyl tRNA is positioned to the A site.

The GTP of the EFTu-GTP complex is hydrolyzed and EFTu is released.

Question 40

What catalyzes peptide bond formation during elongation, and where does the energy for the synthesis come from?

Answer 40

The peptide bond is now formed by nucleophilic attack of the alpha-amino group of the amino acid in the A site on the ester bond between the amino acid and tRNA in the P site. This transferase reaction is catalyzed by the ribosomal RNA! The energy for the synthesis is from the activated amino acid attached to the tRNA.

Question 41

Describe the translocation step in elongation.

• What is involved?
• What does it require?

Answer 41

The final step in elongation is termed translocation. This involves movement of the ribosome one codon on the mRNA with release of the empty tRNA that was present in the P site and transfer of the peptide-bearing tRNA from A to the P site. This step requires elongation factor, EFG and GTP hydrolysis. The A site becomes vacant and the next amino acid charged tRNA enters and binds (by codon/anticodon match), the elongation process is repeated and the protein increased by one amino acid. This continues over and over until the protein is made.

Question 42

How many EF-Tu-GTP amino acyl tRNAs are tried in the A site before the correct one matches?

Answer 42

On average: 10 EF-Tu-GTP amino acyl tRNAs are tried in the A site before the correct one matches! Elongation step occurs in a few milliseconds!

Question 43

What are the termination codons?

Answer 43

Eventually a termination codon in the A site is reached (UAA, UAG or UGA) which no acylated tRNA recognizes.

Question 44

What factors are involved in termination of protein syntesis? What do they do?

Answer 44

Releasing factors RF1, RF2 and RF3 bind to a stop codon and cause the release of the polypeptide chain from the ribosome and the ribosome dissociates and is ready to bind to another message. The releasing factors cause the peptidyl transferase activity to switch to a hydrolase activity to release the completed polypeptide.

Question 45

Why is prokaryotic mRNA polycistronic?

Answer 45

Polycistronic mRNA: In prokaryotes if an initiating codon AUG is not too far from terminal codon, the 70S ribosome will not dissociate but will form a new initiation complex which will lead to translation of the second protein. Thus, one mRNA can code for several proteins and each one might be translated.

Question 46

In E. coli, 30 to 50% of its total energy is needed for what activity?

Answer 46

In E. coli, 30 to 50% of the total energy appears needed for protein synthesis.

Question 47

What are the differences between eukaryotic and prokaryotic protein synthesis initiation?

Answer 47

Initiation: The initiation amino acid in eukaryotes is methionine. Regulation of protein translation may occur in certain instances by phosphorylation of eIF-2 (eukaryotic initiation factor 2). Phosphorylation of eIF-2 inhibits translation.

Question 48

Which have signal peptides, prokaryotes or eukaryotes? What is the signal peptide hypothesis?

Answer 48

Signal peptide: Eukaryotic cells contain both free and membrane bound ribosomes; whereas, prokaryotic cells contain only free ribosomes. The signal for a ribosome to attach to the membrane of the rough ER appears to reside in the protein being synthesized. Signal peptide hypothesis-the signal for a ribosome to attach to the rough ER resides in the first fifteen to thirty amino acids residues at the amino terminal. This is termed the signal peptide.

Question 49

What are the signal peptide steps?

Answer 49

1. SRP: Signal recognition particle binds to the signal peptide just as it produces from the ribosomal complex. SRP blocks translation until the ribosome has docked with the ER membrane. SRP contains 6 proteins and an RNA molecule (300 bases in length) as a central component.
2. SRP receptor: This receptor (found only in the ER) binds SRP when SRP is part if ribosomal complex. Targeting of proteins to the ER is a GTP dependent process. GTP binding to SRP and SRP receptor is required for targeting and GTP hydrolysis is required for regeneration of free SRP and SRP receptor. The SRP receptor forms a gated channel which opens upon ribosome docking and is closed upon ribosome dissociation.
3. Ribophorin: Ribosomal receptor binds the ribosome.
4. Translocation: The signal peptide enters the ER lumen; protein elongation continues; the signal peptide is removed by signal peptidase. ATP is required to transport the protein through the membrane.

Question 50

How many polypeptides are coded by mtRNA?

Answer 50

mtDNA is known to code for nine mitochondrial polypeptides.

a. Cytochrome oxidase -3 of 7 subunits
b. ATPase complex -4 of 9 subunits
c. Cytochrome b complex -1 of 7 subunits
d. Small ribosomal subunit -1 of 22 proteins

Question 51

What is the origin of most mitochondrial proteins?

Answer 51

Transport of Protein into the mitochondria: Most of the mitochondrial proteins are coded for by nuclear DNA and the proteins synthesized in the ER. The N-terminal of proteins targeted for the mitochondria are post-translationally imported into the mitochondria.

Question 52

What are the eukaryotic only protein synthesis inhibitors?

Answer 52

1. Cycloheximide – inhibits peptide bond formation in eukaryotic cells.
2. Diphtheria toxin – Catalyzes the reaction of NAD+ with a eukaryotic elongation factor, resulting in inhibition of translocation

Question 53

What are the eukaryotic/prokaryotic protein synthesis inhibitors?

Answer 53

1. Chloramphenicol – Inhibits peptide bond formation, can inhibit mitochondrial protein synthesis at high levels.
2. Puromycin – Causes premature termination by acting as an aminoacyl-tRNA analogue; the amino acid chain in the P site is transferred to puromycin rather than to the A site.

Question 54

What are the prokaryotic only protein synthesis inhibitors?

Answer 54

1. Tetracycline - Inhibits the binding of aminoacyl tRNAs to the A site on the ribosome; i.e., inhibits elongation.
2. Streptomycin - Bind to the 30S ribosomal subunit and causes dissociation of the translation process; i.e., blocks translation.
3. Erythromycin – Binds to the 50S subunits and inhibits translocation.