BIOMG 3320 LEC 21

What are the five stages of protein synthesis

1. Activation of amino acids (very accurate) || 2. Initiation (involves binding of mRNA and initiator aminoacyl tRNA to small subunit, followed by binding of large subunit) || 3. Elongation (involves synthesis of all peptide bonds, with tRNAs bound to acceptor (A) and peptidyl (P) sites) || 4. Termination and release (occurs when "stop" codon is reached) || 5. Folding and posttranslational processing

How many steps does it take for the initiation complex to form

3 steps

Ex. Bacterial Protein Synthesis, bacteria use the 30S ribosomal subunit

What are the steps of Initiation in Bacterial Protein Synthesis

1. The amino acid is loaded into the 30S subunit || 2. mRNA is loaded into the 30S subunit. The first codon is positioned in the P site and IF2 GTPase recruits the initiation fMet-tRNA || 3. The 50S ribosomal subunit associates, IF2 hydrolyzes GTP to release all initiation factors (IF1, IF2, IF3, & GDP +Pi)

How does mRNA positioning on the ribosome occur?

There are conserved sequences in the 5' UTR of mRNA called the Shine Dalgarno sequence. The purine rich S-D sequence base pairs with the pyrimidine rich 3' end of the 16S ribosomal RNA . This mRNA-ribosomal RNA interaction positions the initiation (5') AUG sequence precisely at the P site on the ribosome

What is IF2

IF2 is a GTPase that binds to the initiation fMet-tRNA, which brings in the fMet-tRNA to bind to the AUG codon in the P site (UAC-AUG base pairing)

What is the initiation fMet-tRNA

This is a specialized initiation tRNA that is modified

Are there other factors that bind to the ribosome?

Yes, there are other initiation factors that bind to different sites of the ribosome

What does Initiator Factor IF1 and IF 2 do

IF1 and IF2 prevent premature assembly of the 30S subunit with the 50S ribosomal subunit

Where does Initiator Factor IF3 and IF1 bind

IF 3 binds in the E site and IF 1 binds in the A site

What is the purpose of the Elongation step in Bacterial protein synthesis?

To synthesize all the peptide bonds with tRNAs bound to aminoacyl A and peptidyl P sites

What occurs during the first elongation step?

Binding of the second aminoacyl-tRNA

What is the mechanism of the first elongation step?

First, EF-Tu recognizes tRNA amino acid in the cytosol and brings it to the ribosome/loads it in the A site. || If the anticodon on the tRNA matches the codon in the A site, then the tRNA will stay and tRNA will dissociate. || EF-Tu hydrolyzes GTP to GDP to release tRNA || EF-TS removes GDP from EF-Tu so that it can be recharged with GTP || This cycle can then repeat

Why does the first step of elongation need energy?

It ensures that the correct amino acid is added since hydrolysis is only triggered when the base pairing between the codon and anticodon match to release tRNA. This mechanism is referred to as ribosomal proofread activity.

How does proofreading on the ribosome occur?

By the GTPase activity of EF-Tu

What occurs during the second elongation step of elongation in bacterial protein synthesis?

Formation of the peptide bond

What is the mechanism of the second step of elongation in bacterial protein synthesis?

1. The amino group is activated in the ribosome to form a nucleophile and attack the previous amino acid. || 2. This converts the ester bond, which undergoes hydrolysis, to become a super stable peptide bond || 3. After the bond is formed, it will tip away from the A site and move into the P and E site. This is called peptidyl transfer and must be aided by another ribosomal RNA

What is peptidyl transferase?

A 23S rRNA ribozyme that allows the tipping away from the A site and movement into the P and E site of the ribosome???

What occurs during the third step of elongation in bacterial protein synthesis?

Translocation

What is the mechanism of the third step of elongation in bacterial protein synthesis?

1. EF-G-GTP binds to tRNA in A site || 2. The structure of EF-G mimics the structure of EF-Tu complexed with tRNA so that it can insert into the A site || 3. After it inserts, it hydrolyzes GTP to induce a conformational change in the ribosome to move forward. || 4. This leaves the A site open to the third incoming aminoacyl-tRNA

What does the E site stand for?

The exit site

What does the P site stand for?

The peptidyl site

What does the A site stand for?

The aminoacyl site

When does termination and release occur

When the "stop" codon is reached

Where is the stop codon encountered?

At the A site

Describe what occurs during Termination of Protein synthesis

The Stop codon is encountered at the A site || Release factors bind || Peptidyl transferase now acts as a hydrolase, transferring the peptide to H2O, thus releasing peptide from tRNA || It is Aided by the ribosomal recycling factors (RRF), IF3, and energy provided by EF-G mediated GTP hydrolysis || Uncharged tRNA dissociates and release factors and expelled || IF 3 prevents the association of 50S and 30S ribosomes

What does peptidyl transferase act as during termination of protein synthesis

Acts as a hydrolase by transferring the peptide to H2O which releases the peptide from the tRNA

What is termination of protein synthesis aided by?

It is aided by the ribosomal recycling factors (RRF), IF3, and energy provided by EF-G mediated GTP hydrolysis

How many GTP molecules are hydrolyzed during the elongation stage of protein synthesis(pollev)

2

Does the formation of the initiation complex in bacteria need energy input from GTP hydrolysis (pollev)

Yes?

Is the formation of peptide bonds catalyzed by a protein subunit on the ribosome? (pollev)

No? It is catalyzed by peptidyl transferase a 23S ribozyme?

Does the termination of protein translation need any energy input?( pollev)

Yes, it needs energy provided by EF-G mediated GTP hydrolysis

What steps in Protein synthesis require energy input????

Termination || Initiation || Elongation? || All steps????

When does translation take place in bacteria?

While transcription is still going on

Does translation take place while transcription is still going on in eukaryotes?

No, translation and transcription are NOT coupled in eukaryotes

What is a polysome?

Multiple ribosomes bound to RNA and making protein at the same time

What disrupts protein synthesis

Antibiotic puromycin

How does antibiotic puromycin disrupt protein synthesis?

Puromycin resembles the aminoacyl end of a charged tRNA || It binds to the A site of peptidyl transferase and participates in peptide bond formation || It is a "terminator" for peptide chain elongation

What is puromycin in peptide chain elongation

It is "terminator" for peptide chain elongation

What does puromycin resemble

The aminoacyl end of a charged tRNA

Where does puromycin bind

To the A site of peptidyl transferase

What does puromycin participate in after binding to the A site of peptidyl transferase?

Puromycin participates in peptide bond formation

What does Puromycin only inhibit in prokaryotes but not in eukaryotes? And why is this important

Puromycin only inhibits protein synthesis in prokaryotes but not in eukaryotes. As such, this is why it can be given to humans as an antibiotic

What are some other antibiotics that inhibit protein synthesis?

Tetracycline, chloramphenicol, cyclohexamide, and streptomycin

Do Eukaryotic mRNAs have the Shine-Delgano sequence?

No, there is no equivalent of the SD sequence in eukaryotic mRNAs. The first AUG from the 5' cap is the start codon

What is the structure of mRNA in the Eukaryotic initiation complex?

It is circulized

What is mRNA processed to become and what does it have?

It is processed to make mature mRNA and has a 5' cap and poly A tail

How does mRNA become circularized?

In the cytosol, Cap binding proteins, and poly A binding protein (PABP) bind mRNA and form the mRNA into a circular structure??

How is translation initiated in eukaryotic mRNA initiation?

The 40S small subunit of eukaryotic ribosome binds to the PABP/cap protein complex and slides along until it finds the first AUG and initiates translation. The 60S large subunit comes in to assemble the full ribosome

Do mature mRNAs travel long distances? If so, what can this result in?

Yes, mature mRNAs travel long distances from the nucleus to the cytoplasm and may be subjected to nuclease degradation

What are the only types of mRNAs that can be translated?

Only intact mRNAs are translated. I.e only mRNA that are capped at the 5' end and polyadenylated at the 3' end will be translated.

How is the circularized mRNA linked to the 40S ribosome?

elF4E binds to the 5' cap and elF4G binds to the poly (A) binding protein (PABP) at the 3' end of the mRNA. The elF4G also binds to elF3, linking the circularized mRNA to the 40S ribosome.

What is the function of the Shin Dalgarno sequence

It is located at the 5' end of the mRNA. This sequence binds to the 16S rRNA of the small ribosomal subunit which will position the AUG(start site) at teh P site

How is the initiation complex formed

First, the small ribosomal subunit binds IF1 at the A site and IF 3 at the E site

Second, the mRNA comes and binds to the small ribosomal subunit, and with the help of the Shine Dalgarno sequence, it will bind the mRNA to the 16S rRNA of the ribosome, which will result in positioning the AUG at the P site

Third, the IF2 factor bound to GTP will bind to the subunit with the initiator fmet tRNA.

Fourth, the large ribosomal subunit will associate, and IF2 bound to GTP will dissociate to GDP + Pi and all initiating factors will dissociate

The fmet-TRNA will stay at the P site but the E site and A site will be empty and ready for elongation

What occurs during elongation to allow for incorrect base pairs to dissociate?

A lag caused by EF TU GTP hydrolyzing

Does teh ribosome proofread what amino acid youre putting directly?

No, it can only proofread how well that tRNA is base pairihng to the mRNA

I.e. if the tRNA is correctl paired to the right amino acid, then everything will be fine. But the ribosome can't tell if the wrong amino acid is on a tRNA; it only knows that the tRNA is binding to the mRNA properly

What does EF TS do in the first step of elongation

It allows EF TU GDP to be recycled to EF Tu GTP

Does the peptide bond formation in elongation step 2 take additional energy

No, we have been investing energy through GTP hydrolysis

What catalyzes the formation of the peptide bond in elongation step 2

The rRNA in the large subunit

What does hydrolysis of EF G-GTP (translocase) do?

It moves the ribosome

What are the three steps of elongation in bacterial peptide synthesis

1.) Elongation?

1. The next aminoacyl tRNA comes into the A site, bound to EF-Tu-GTP

2. EF-Tu-GTP will hydrolyze the GTP which will create a lag. This lag will allow for incorrectly base paired tRNAs to dissociation (proofreading)

3. The EF-Tu-GDP will dissociate and EF-Tu-GTP will be recycled by EF-Ts

2.) Accommodation: making room to form the peptide bonds

1. The aminoacyl tRNA in the A site will change its formation to be closer to the P site tRNA to allow for peptide bond formation

2. The rRNA in the large subunit will catalyze peptide bond formation (no additional energy required)

(amino group attacks at the carboxylic carbon, leading to a tRNA without amino group and an aminoacyl tRNA with a growing polypeptide chain

3. Translocation, moving from ribosome

1. EF G GTP (translocase) bids to the A site and hydrolyzes its GTP

2. Pi and EF-G-GDP dissociate

2. This hydrolysis moves the ribosome so that the empty tRNA is now in the E site, the aminoacyl tRNA with the growing polypeptide is in the P site,and the A site is empty to allow for a new aminoacyl tRNA to come in

How does termination of bacterial peptide synthesis occur?

1. A stop codon in the A site will signal termination of synthesis

2. A termination factor(similar in structure to tRNA) will bind at the A site

3. The Termination Factor will then hydrolyze the bond between the polypeptide and the tRNA in the P site.

4.) The EF-G-GTP (translocase) will translocate the ribosome through hydrolysis, which will lead to the TF being in the A site and the ribosome will dissociate from the mRNA