Biochem 432: Proteins

Question 1

Which of the following is true?

1. tRNAs are the adaptor molecules between amino acids and mRNA
2. Protein synthesis doesn’t occur in ribosomes
3. The third base in codons are equally important as the first and second base in binding to tRNA
4. Each codon ONLY codes for one amino acid and one amino acid

Answer 1

TRUE: 1. tRNAs are the adaptor molecules between amino acids and mRNA

FALSE: Protein synthesis doesn’t occur in ribosomes
—-> Ribosomes synthesize proteins

FALSE: The third base in codons are equally important as the first and second base in binding to tRNA

FALSE: Each codon ONLY codes for one amino acid and one amino acid
—-> Each codon codes for a specific amino acid but multiple codons can code for the same amino acid

Question 2

What are the five stages of protein synthesis?

Answer 2

1. Activation of amino acids: tRNA aminoacylated. Synthetase. ATP. Mg2+.
2. Initiation of translation: mRNA and aminoacylated tRNA bind to ribosome. Mg2+. GTP.
3. Elongation: cycles of aminacyl-tRNA binding and peptide bond formation until a STOP codon is reached. Mg2+. GTP
4. Termination and ribosome recycling: mRNA and protein dissociate, ribosome recycles
5. Folding and posttranslational processing: catalyzed by a variety of enzymes

Question 3

Name three early advances in understanding protein synthesis

Answer 3

Proteins synthesized at ribosomes

tRNA acts as an “adapter” to translate mRNA into protein

Amino acids activated for synthesis by attachment to tRNA via aminoacyl-tRNA synthetases

Question 4

Describe the ribosome

Answer 4

—65% rRNA and 35% protein
—two subunits (30S and 50S) in bacteria
—Prokaryotic ribosomes have 3 tRNA binding sites
—Eukaryotic ribosomes are larger (80S)
—Folding pattern of rRNAs are highly conserved
—Found in the cytosol, mitochondria and chloroplasts of all cells
—Bind and orient mRNAs and aminoacyl-tRNAs
—Organize interactions between codons and anticodons in aminoacyl-tRNAs
—Catalyze formation of peptide bonds between adjacent amino acid residues - RNA does the catalysis of peptide bond formation
—Move along mRNAs and synthesize proteins

Question 5

Describe tRNA as an adaptor molecule

Answer 5

—provides a physical connection between the DNA-RNA genetic code and amino acids
—the driving force for protein synthesis
—used to recognize codons in mRNA
—cloverleaf in structure
—Most contain G at 5’ ends
—ALL have CCA at 3’ end
—Contain modified bases: Inosine (I), pseudouridine (y), dihydrouridine (D), ribothymidine (T), and methylated bases (mG, mI), and so on
—Contain an amino acid arm, anticodon arm, D arm, dihydrouridine (D)

Question 6

Explain molecular recognition of codons in mRNA by tRNA

Answer 6

—The codon sequence is complementary with the anticodon sequence
—The codon in mRNA base pairs with the anticodon in tRNA via hydrogen bonding
—The alignment of two RNA segments is antiparallel

Question 9

Describe the Wobble hypothesis

Answer 9

— Proposed by Crick
— Base pairing rules may be realized at the third position of the codon (first position of the anticodon)
— Inosine is formed by adenosine deamination paired with A, C and U.
— H-bonds between I & A,C,U are weaker and therefore were nickname “wobble” base pairs

Question 9

Describe aminoacylation of tRNA (Stage 1 of protein synthesis)

Answer 9

Charge tRNAs = Activation of amino acids

The function of aminoacyl-tRNA synthetase enzymes is to charge tRNA molecules with the correct amino acid

1. Creation of aminoacyl intermediate: COO- of amino acid attacks phosphate of ATP to create an aminoacyladenylate intermediate.

Phosphate (PPi) is also cleaved so the reaction is driven forward by two phosphoanhydrive bond cleavages

2. Transfer of amino acyl to tRNA via Aminoacyl-tRNA syntehtases which transfer aminoacyl groups from ezymes to tRNA

2’-OH or 3’-OH of the last nucleotide at the 3’ end of the tRNA attacks phosphat of aminoacyl intermediate to create a phosphodiester bond between amino acids and tRNA

Thermodynamically favorable reaction

Question 10

Describe Aminoacyl-tRNA synthetase

Answer 10

Enzyme required to catalyze the attachment of a specific amino acid to the 3’ acceptor stem of the matching tRNA molecule

Each amino acid has its own aminoacyl-tRNA synthetase

The correct amino acids are chosen in a two-step process. Most aminoacyl-tRNA synthetases have an aminoacylation site and an editing site.

Question 11

Describe the prokaryotic stage 2 of protein synthesis, initiation

Answer 11

Initiation: binding of mRNA and initiator aminoacyl-tRNA to a small subunit, followed by binding of a large subunit

The first tRNA is unique.

The first codon of any peptide is AUG (Met).

All organisms have two tRNAs for Met.

In bacteria, tRNAfMet is charged with N-formylmethionine (fMet) for the 5’ AUG initial codon; Met in an internal position is added with normal tRNAMet.

Requires: 30S ribosomal subunit, mRNA, fMet-tRNA, initiation factors, GTP, 50S ribosomal subunit, Mg2+

Step 1: The 30S ribosomal subunit binds IF-1, IF-3, and mRNA.
Step 2: GTP-bound IF2 and fMet-tRNAfMet joins the complex.
—Formylmethionine tRNA binds to the peptidyl (P) site along with initiating(5’)AUG.
—All other Aminoacyl-tRNA bind to the aminoacyl (A) site.
Step 3: 50S subunit associates

Question 12

Give a summary of prokaryotic initiation

Answer 12

The AUG at the start of an open reading frame is identified by initiation factors (IFs), the ribosome, and a special initiator methionine tRNA.

The SD sequence upstream of AUG interacts with 16s rRNA, and the interaction is important to determine the AUG orientation.

This results in a ribosome with a methionine-loaded tRNA bound in the P site.

Early initiation involves the small ribosomal subunit, and then the large subunit joins the complex.

The ribosome is now ready to move along the mRNA. The mRNA is read 5’ to 3’.

Question 13

Explain Eukaryotic Initiation

Answer 13

1. Binding eIF2 and Met-tRNA to 40S to form 43S;
2. eIF3 binds to the eIF4G subunit of the 5’ cap binding complex; eIF4G binds to PABP to form a circularized mRNA; eIF4A is an ATPase and RNA helicase;
3. Ribosomal scanning in the 5’ to 3’ direction on the mRNA to the first AUG codon (sometimes not the first AUG codon).

Initiation usually occurs at the first AUG in the mRNA, but this is sometimes inefficient and more initiation occurs at the second or third AUG.

There are no eukaryotic equivalents of the Shine-Dalgarno sequences. Kozak sequence containing AUG in many eukaryotic mRNAs.

Uses more initiation factors - The 40S ribosomal subunit, bound to a number of initiation factors (over 12) including eIF1A and eIF3 (functional homologs of IF-1 and IF-3), is primed to scan the mRNA (pre-initiation complex).

The eukaryotic mRNA 5′ cap and 3′ poly(A) tail are also involved in initiation, preparing the mRNA to be scanned by the ribosome. Has a step that circularizes the mRNA during initiation. This forms a closed loop complex, and may function as a quality control to weed out unfinished or damaged mRNAs

Question 14

What are the differences between initiation in prokaryotes and eukaryotes?

Answer 14

Eukaryotic
— Uses 40S and 60S subunits to form the 80S ribosome
— More initiation factors are involved
— Requires specific mRNA sequences at both the 5′ and 3′ ends of the mRNA to form circularized mRNA for scanning.
— Recognition of the AUG is sensitive to the sequence context – the Kozak sequence (consensus: (A/GXXAUGG)

Prokaryotic
— Uses 30S and 50S subunits to form the 70S ribosome
— Three initiation factors involved
— Shine-Dalgarno sequence appears 5-13 upstream of the first AUG codon to orient mRNA within the ribosome.

Question 15

Explain the third stage of protein synthesis, elongation

Answer 15

Elongation: synthesis of all peptide bonds - with tRNAs bound to aminoacyl (A) and peptidyl (P) sites

Step 1: binding of the incoming aminoacyl-tRNA
—Incoming aminoacyl-tRNA binds first to an EF-Tu –GTP complex.
—The aminoacyl-EF-Tu-GTP complex binds to the aminoacyl (A) site of the 70S initiation complex.
Step 2: Peptide bond formation
—two amino acids bound to tRNAs positioned for joining; one on the A site and the other on the P site
—Reaction is catalyzed by the 23S rRNA
Step 3: Translocation of the ribosome
—ribisome moves one condon toward the 3’-end of the mRNA using energy via GTP hydrolysis

Question 16

Describe peptidyl transferase reaction

Answer 16

Peptidyl transferase is 23S rRNA!!!!

Creates the peptide bond that holds the polypeptide together

Amino group of the amino acid attached to the 3′ terminus of the tRNA in the A site attacks the carbonyl carbon of the peptidyl-tRNA in the P site, resulting in an extended A-site peptidyl-tRNA.

Question 17

Explain termination, stage 4 of protein synthesis

Answer 17

Termination is also similar in prokaryotes and eukaryotes; signaled by a stop codon;

Stop codon (UAA, UAG, or UGA) enters the A site;

Release factor hydrolyze GTP to promote disassembly of ribosomal complex

Question 18

Some posttranslation modifications include:

Answer 18

enzymatic removal of formyl group from first residue, or removal of Met and sometimes additional residues

Removal of signal sequences or other regions

Removing sequence to activate an enzyme

Proteolysis, disulfide bond formation

Attachment of a functional group or macromolecule to the fully synthesized protein

Question 19

What are six common examples of attachments of a functional group in eukaryotic cells in posttranslational modifications?

Answer 19

1. (de)phosphorylation
2. Methylation
3. Acetylation
4. Ubiquitination (designates proteins that need to be degraded)
5. Glycosylation
6. Lipid modification

Question 20

Which amino acids are phosphorylated in posttranslation modification?

Answer 20

Tyrosine, serine and threonine —> amino acids that contain hydroxyl groups becase the hydroxyl groups allow for phosphorylation via KINASE

Amp attachment to a molecule affects some reactions via adenylation

Question 21

Which amino acid gets methylated?

Answer 21

Lysine because it has a second amine group (Replace an H in the NH3 group with a methyl group)

methyltransferase (methylates)
Amine oxidase (demethylates)

Question 22

Which amino acids can get acetylated? What are the enzymes?

Answer 22

Lysine gets actyelated

Histone acetyltransferase + Acetyl-CoA: acteylateds Lysine

Histone deacetylase + H2O: deacetylates Lysine

Question 22

Which amino acids are ubiquinated?

Answer 22

Lysine

Question 23

What are three common lipid modifications?

Answer 23

1. Prenylation: addition of isoprenoid group
   — CaaX motif: “C” is cysteine, ‘a’ is any aliphatic amino acid, “X” is the C-terminal residue
2. Myristoylation: the attachment of a saturated 14-carbon fatty acid to the N terminus glycine residue of target protein
3. Palmitoylation: the attachment of a saturated 16-carbon fatty acid on the sulfur atom of internal cysteine residues found anywhere in the protein

Question 24

Describe ER signal peptide sequence

Answer 24

Peptide sequence that targets ribosomal complexes for transport to the rough ER

Depends on the presence of a specific amino acid sequence at the N terminus of the nascent polypeptide

Important for the vesicle-based transportation of proteins

ER-targeted proteins contain signal peptide sequences with four functional regions

Question 25

Explain Cotranslational translocation

Answer 25

Step 1: N-terminal ER signal sequence (SS) emerges from the ribosome first during nascent protein synthesis.

Step 2: Signal recognition particle (SRP) binds SS – arrests protein synthesis

Step 3: SRP-nascent polypeptide chain–ribosome complex –
Binds to the SRP receptor in the ER membrane
Interaction is strengthened by the binding of GTP to both the SRP and its receptor.

Step 4: Transfer of the nascent polypeptide–ribosome to the translocon –
Opens translocation channel to admit the growing polypeptide
Signal sequence – transferred to a hydrophobic binding site next to the central pore
SRP and SRP receptor – hydrolyze bound GTP; dissociates SRP from ribosome and receptor; restarts protein synthesis (can initiate the insertion of another polypeptide chain)

Step 5: Elongating polypeptide chain
Passes through the translocon channel into the ER lumen
Signal sequence – cleaved by signal peptidase and rapidly degraded

Step 6: Growing peptide chain – continues extrusion through the translocon into the ER as the mRNA is translated toward the 3′ end

Step 7: Translation completes at mRNA stop codon – ribosome is released

Step 8:
Nascent protein – remainder drawn into the ER lumen and folds into native conformation
Translocon closes.

Question 26

Describe nuclear import and export of eukaryotic proteins

Answer 26

Nuclear functions require a variety of proteins, all of which are synthesized in the cytosol.

These proteins are imported to the nucleus and then exported to the cytosol to be recycled.

Nuclear import requires a stretch of basic amino acids (nuclear localization signal).
— Must be exposed on the protein’s surface
— Importins and exportins aid active transport.
— Some exportins recognize nuclear export signal or RNA sequences.

Question 27

What are three important purposes of protein synthesis?

Answer 27

1. Unraveling the mechanism of protein synthesis
2. Effective antibiotics - affect prokaryotic but not eukaryotic protein synthesis
3. Engineer protein synthesis

Question 28

What anitbodies can prohibit protein synthesis in eukaryotes

Answer 28

Chloramphenicol, Erythromycin, Tetracyclines, Streptomycin

Question 29

Name 4 major biochemical targets in bacteria

Answer 29

1. Inhibits bacterial wall synthesis (cillins)
2. Block protein synthesis
3. Block DNA replication
4. Target cell membrane

Question 30

Eukaryote-specific antibiotics: Cycloheximide

Answer 30

blocks peptidyl transferase of 80S ribosomes, not 70S

Question 31

Eukaryote-specific antibiotics: Diphtheria Toxin

Answer 31

• An NAD+-dependent ADP ribosylase; catalyzes ADP-ribosylation of eEF2 and inactivating it. One toxin molecule ADP-ribosylates many eEF-2s, so just a little is lethal!

Question 32

Eukaryote-specific antibiotics: Ricin

Answer 32

One of the most toxic substances known!!!! It is from castor bean, the very poisonous seed of the castor-oil plant. It has two functionally different polypeptide chains, A and B, which are disulfide-linked. B subunit mediates endocytosis. Endocytosis followed by disulfide reduction releases A subunit, which attaches to 28S ribosomal RNA in the 60S ribosomal subunit and inhibits protein elongation.

Question 33

Puromycin

Answer 33

inhibits both prokaryotic and eukaryotic protein synthesis

Question 34

Which of the following is false?

1. Penicillin inhibits bacterial wall synthesis, and puromycin terminates translation in both prokaryotes and eukaryotes
   
2. Lysine residues in proteins can be methylated, acetylated, or ubiquitinated
   
3. Ubiquitination required three types of enzymes E1, E2, and E3, and they are ubiquitin-activating enzyme, conjugating enzyme, and ligase enzyme respectively
4. Ubiquitination involves a lysine residue of the target protein, and a C-terminus glycine residue of ubiquitin. The alpha amino group of lysine residue forms a peptide bond with the glycine residue.

Answer 34

4. Ubiquitination involves a lysine residue of the target protein, and a C-terminus glycine residue of ubiquitin. The alpha amino group of lysine residue forms a peptide bond with the glycine residue.