Biochem 2 lect 8- Protein synthesis and degradation

What is the function of ribosomes?

To assemble amino acids into proteins.

What are the two subunits that make up a ribosome?

The small subunit and the large subunit.

What is the role of tRNA in protein synthesis?

It carries amino acids to the ribosome.

What is the start codon in protein synthesis?

AUG (methionine).

How does protein synthesis differ in eukaryotes compared to prokaryotes?

Eukaryotes have a more complex process due to compartmentalization.

What are some examples of inhibitors of protein synthesis?

Tetracycline, streptomycin, chloramphenicol.

What is the function of chaperone proteins in protein folding?

To assist in the correct folding of other proteins.

What is the proteasome responsible for in protein degradation?

Breaking down unneeded or damaged proteins.

How many subunits does the E. coli ribosome consist of?

Two unequal subunits.

What is the mass of the 30S subunit of the E. coli ribosome?

930 kD.

Which rRNAs are present in the 50S subunit of the E. coli ribosome?

23S rRNA and 5S rRNA.

How many copies of each protein are there per ribosome, except L7/L12?

One copy.

How many co-copies does L7/L12 have?

4 co-copies.

What is the difference between the L7/L12 proteins?

The extent of acetylation at the N-terminus.

Which protein is common to large and small subunits?

S20 = L26.

What is required for functional ribosomes to assemble?

Mixing individual proteins and rRNAs.

What is known about the high resolution structures of ribosome subunits?

Both large and small subunits have known high resolution structures.

What runs through the large subunit of the ribosome?

A tunnel.

What is thought to thread through the tunnel during protein synthesis?

The growing peptide chain.

What are the two main components of a ribosome?

Small subunit and large subunit.

What is the function of the ribosome's small subunit?

It helps to locate the start codon on the mRNA.

What is the function of the ribosome's large subunit?

It catalyzes the formation of the peptide bonds between amino acids.

What is the supposed origin of mitochondrial and chloroplast ribosomes?

Prokaryotic origin.

Where does ribosome biogenesis occur?

In nucleolus and cytoplasm.

How do the ribosomes of higher eukaryotes compare to those of lower eukaryotes?

More complicated.

What are the three phases of protein synthesis?

Initiation, elongation, termination.

What is involved in the initiation phase of protein synthesis?

Binding of mRNA and initiator aminoacyl-tRNA to the small subunit, followed by binding of the large subunit.

What occurs during the elongation phase of protein synthesis?

Synthesis of all peptide bonds with tRNAs bound to acceptor (A) and peptidyl (P) sites.

When does termination occur in protein synthesis?

When the 'stop codon' is reached.

What does the A site stand for in protein synthesis?

Acceptor site.

What does the P site represent in protein synthesis?

Peptidyl site.

What is the function of the E site in protein synthesis?

Exit site.

What are the components required for peptide chain initiation in prokaryotes?

mRNA, the 30S and 50S ribosomal subunits, initiation factors, GTP, and a specific charged tRNA (f-Met-tRNA f Met).

What is the initiator tRNA in prokaryotic initiation?

f-Met-tRNA (formylated methionine).

When is f-Met-tRNA used?

Only for initiation.

Which tRNA is used for regular methionine addition in prokaryotic initiation?

Met-tRNA (methionine).

What is the first amino acid of all E.coli proteins?

N-formyl methionine.

Is the formyl group cleaved off from N-formyl methionine in E.coli proteins?

Yes, in about half of the proteins.

Who adds the formyl group to methionine?

A formyl transferase.

How many GC base pairs are present in the f-Met-tRNA f Met?

What is required for the correct registration of mRNA on the ribosome during initiation?

Alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of prokaryotic mRNAs.

What is the purine-rich segment on the 5'-end of prokaryotic mRNAs known as?

The Shine-Dalgarno sequence.

What components form the 30S initiation complex?

Initiation factor proteins, GTP, N-formyl-Met-tRNA-fMet, mRNA, and 30S ribosome.

What are Shine-Dalgarno sequences?

Short nucleotide sequences found in the bacterial mRNA upstream of the start codon.

Where are Shine-Dalgarno sequences found?

In the bacterial mRNA upstream of the start codon.

What is the function of Shine-Dalgarno sequences?

They help in the initiation of protein synthesis in prokaryotes.

What components are required for peptide chain elongation in prokaryotes?

mRNA, 70S ribosome, peptidyl-tRNA complex, aminoacyl-tRNAs, elongation factors.

What is the role of mRNA in peptide chain elongation in prokaryotes?

It is required for peptide chain elongation.

What is the role of 70S ribosome in peptide chain elongation in prokaryotes?

It is required for peptide chain elongation.

What is the role of peptidyl-tRNA complex in peptide chain elongation in prokaryotes?

It is required for peptide chain elongation.

What are aminoacyl-tRNAs in the context of peptide chain elongation in prokaryotes?

They are required for peptide chain elongation.

What are elongation factors in the context of peptide chain elongation in prokaryotes?

They are a set of proteins required for peptide chain elongation.

What is the percentage of EF-Tu in total protein in E. coli?

5%.

What does EF-Tu bind to?

Aminoacyl-tRNA and GTP.

Where does aminoacyl-tRNA bind in the ribosome?

A site, as a complex with 2 EF-Tu and 2 GTP.

What happens after GTP is hydrolyzed?

EF-Tu:GDP complexes dissociate.

How does EF-Ts recycle EF-Tu?

By exchanging GTP for GDP.

What is the peptidyl transfer reaction?

It is the process of transferring the peptide chain from the tRNA in the A site to the tRNA in the P site during protein synthesis.

What is the central reaction of protein synthesis?

Peptidyl Transferase.

Which component is the peptidyl transferase in protein synthesis?

The 23S rRNA.

What is the significance of the bases in the 'reaction center' of 23S rRNA?

They are highly conserved in all of biology.

What follows the translocation of peptidyl-tRNA from the A site to the P site?

Translocation of peptidyl-tRNA from the A site to the P site follows.

How many GTPs are hydrolyzed for each amino acid incorporated into a peptide?

Three GTPs.

What drives essential conformation changes in protein synthesis?

Hydrolysis.

How many high-energy phosphate bonds are expended per amino acid residue added?

Five (three GTP and two in amino acid activation).

Where are the two high-energy phosphate bonds expended in amino acid activation?

In amino acid activation via aminoacyl-tRNA synthesis.

What do release factors recognize in the A site during peptide chain termination?

The stop codon.

What happens when release factors with a nonsense codon are present at the A site?

The peptidyl transferase transforms into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier.

What is the process of ribosome assembly and disassembly known as?

Ribosome life cycle.

What are the stages involved in the ribosome life cycle?

Assembly, maturation, and disassembly.

During which stage of the ribosome life cycle are ribosomal proteins and RNA assembled into ribosomal subunits?

Assembly stage.

What happens during the maturation stage of the ribosome life cycle?

Ribosomal subunits are processed and modified to become functional ribosomes.

At which stage of the ribosome life cycle do ribosomes disassemble into their constituent proteins and RNA?

Disassembly stage.

What does EM of polysomes show?

Multiple ribosomes translating the same mRNA.

What is happening to the ribosome in EM of polysomes?

The ribosome is growing a peptide chain.

In prokaryotes, what occurs simultaneously?

Transcription and translation.

What are the two important features of eukaryotic mRNA?

5'-methyl-GTP cap and the poly A tail.

How many eukaryotic initiation factors are involved in the initiation of protein synthesis in eukaryotes?

At least 11.

What is the special characteristic of the initiator tRNA in eukaryotes?

It carries only Met and functions only in initiation, and it is called tRNA i Met but it is not formylated.

What is the function of the sequence with no Shine-Dalgarno sequence?

Required for ribosomal binding and enhances the stability of the mRNA.

What does the sequence with no Shine-Dalgarno sequence enhance?

Stability and translational efficiency.

What is the first step of eukaryotic initiation?

Formation of ternary complex of eIF-2, GTP and Met-tRNA i Met.

What does the ternary complex of eIF-2, GTP and Met-tRNA i Met bind to?

40S ribosomal subunit:eIF-3:eIF4C complex to form the 40S pre-initiation complex.

At what stage is there no mRNA and no codon association with Met-tRNA i Met?

40S pre-initiation complex.

What is required for the formation of the 40S initiation complex?

mRNA and several other factors.

What is required for the formation of the 40S initiation complex?

ATP.

What does the PolyA binding protein bind to?

PolyA tail of mRNA.

What is the function of cap binding protein?

It binds to the 5' cap of mRNA to initiate translation.

Where is the protein coding region located in mRNA?

Between the 5' cap and the 3' PolyA tail.

What is the key process in the regulation of initiation?

Phosphorylation.

Which proteins involved in initiation are activated by phosphorylation?

Ribosomal protein S6 and eIF-4F.

What happens when eIF-2a is phosphorylated?

It binds all available eIF-2B and sequesters it.

What are two important purposes of inhibitors of protein synthesis to biochemists?

1. Unraveling the mechanism of protein synthesis. 2. Acting as effective antibiotics by affecting prokaryotic but not eukaryotic protein synthesis.

How does streptomycin, an aminoglycoside antibiotic, affect protein synthesis?

It induces mRNA misreading, resulting in mutant proteins that slow the rate of bacterial growth.

What is the effect of puromycin on protein synthesis?

It binds at the A site of both prokaryotic and eukaryotic ribosomes, accepts the peptide chain from the P site, and terminates protein synthesis.

What is the target of Diphtheria Toxin enzyme?

EF-2.

What does EF-2 have?

Diphthamide.

What effect does toxin-mediated ADP-ribosylation of EF-2 have on its activity in protein synthesis?

It allows it to bind GTP but makes it inactive.

How many EF-2s does one toxin molecule ADP-ribosylate?

Many EF-2s.

What type of substance is ricin considered to be?

One of the most deadly substances known.