Week 11 Ribosomes

What are the similarities and differences between eukaryotic and prokaryotic ribosomes?

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Ribosomes translate mRNA in the , synthesizing the corresponding protein from _

5' to 3' direction, N-terminus to C-terminus

Where is the first codon found in prokaryotic mRNA?

Approximately 25 nucleotides downstream from the transcript's 5' end

Where does the ribosome bind to the prokaryotic mRNA to recognize the first codon?

Binds about 10 nucleotides upstream of the initiation site at purine rich consensus sequence called Shine Dalgarno (SD) sequence

How has the presence of SD site been found?

Foot-printing experiments

The SD sequence is first recognized by

3' end of the 16S rRNA (in the small ribosomal subunit)

What happens if there's a mutation in the 16S rRNA?

Disrupts base painting, inaccurate identification of the mRNAs start site, wrong reading frame

The AUG start codon that follows the SD sequence signals for the incorporation of

Formyl methionine (fMet) residue

The fMet amino acid is carried exclusively by

Initiator tRNA (tRNAi fMet)

Where does the initiation codon attach to/position in the ribosome?

P site (NOT A SITE)

How does charging of tRNAifMet with formyl methionine occur?

First, unmodified methionine is added/tRNAifMet is charged with normal methionine. Then, enzyme modifies it to N-formyl-Met

Why is modification of methionine to N-formyl-Met important?

Additional formyl group mimics a peptide bond, allows the charged tRNA to bind to P site and not A site

What subunit h-bonds with the mRNA transcript for correct reading frame for translation?

The 16S rRNA in the 30S subunit

The delivery of aminoacyl-tRNA to the A site of the ribosome is facilitated by

EF-Tu

What facilitates the release of GDP and the binding of a new GTP molecule to EF-Tu?

EF-T

Peptide bond formation takes place at the

P site, at the large ribosomal subunit

The acceptor arm of the tRNA carrying the growing polypeptide chain moves into the

P site

The deacylated tRNA acceptor arm moves to the

E site

What is the GTP-bound elongation factor?

EF-G

What does the EF-G do in order for translocation to take place?

Hydrolyzes GTP into GDP, this moves the anticodons of the tRNAs one step to the left

The structure of EF-G mimics

EF-Tu bound amino-acyl tRNA complex, so it fits nicely near A-site

Where does EF-G attach?

Attaches to the 50S large ribosomal subunit, near the A site

Multiple ribosomes can be cycling through the translation elongation process on the same mRNA transcript, at the same time, in groupings called

Polysomes

How can polysomes be visualized?

Electron microscopy

Protein synthesis is independent of

The amino acid charged to a particular tRNA molecule, only depends on pairing between mRNA codon and tRNA anticodon

The sulfur of the cysteine can then be converted into a hydrogen atom using

Raney nickel, creating Ala-tRNACys, a mischarged tRNA

During translation, the first two nucleotides of an mRNA codon must form

Strict Watson Crick base pairs, third position has greater steric freedom and allows for both Hoogsteen and WC base pairing between the tRNA and mRNA

A tRNA with the anticodon 5'-IAC-3' can recognize mRNA codons

5'-GCU-3', 5'-GCC-3', and 5'-GCA-3'

What part of the ribosome allows for flexibility in the third position?

16S rRNA, only proofreads the first 2 positions of the codon before tRNA attaches

The 16S rRNA features three conserved nucleotides that hydrogen bond with

The first and second position on the codon, the second and third position on the anticodon (REMEMBER THE NUMBERING THO), the third position of the codon can be whatever lolz

When STOP codons are encountered at the A site of the ribosome, they are instead recognized by

Release factors (RFs), RF-1 or RF-2 will bind to it, assisted by GTP-bound RF-3. RF-1 is for UAA and UAG, RF-2 is for UAA and UGA

What is the role of the ribosomal release factor (RRF)?

Binds to the ribosome and triggers the release of the entire translation complex, using energy from the hydrolysis of another molecule of GTP

How many ATPs and GTPs would be consumed in the synthesis of a 9 amino acid polypeptide chain?

18 ATPs and 19 GTPs are required to synthesize a 9 amino acid protein

What is the key difference between eukaryotic and prokaryotic translation initiation?

Eukaryotic initiation is more complex and involves more protein factors than prokaryotic initiation

What two major complexes form independently at the start of eukaryotic translation initiation?

eIF4F complex (binds 5' cap of mRNA) and 43S pre-initiation complex (contains small ribosomal subunit + Met-tRNAiMet + eIFs like eIF2-GTP)

What are the components of the eIF4F complex?

eIF4E (binds 7-methyl-G cap), eIF4G (scaffold protein), and other components like eIF4A (a helicase)

What does eIF4E specifically bind to?

The 7-methyl-G cap at the 5' end of eukaryotic mRNA

What does the 43S pre-initiation complex contain?

Small (40S) ribosomal subunit, initiator tRNA (Met-tRNAiMet), and eIF2-GTP

What is the role of eIF2 in the 43S complex?

It binds GTP and helps load Met-tRNAiMet onto the small ribosomal subunit

How is the initiator methionine tRNA in eukaryotes different from the one used internally?

It is a distinct tRNA (tRNAiMet) used only for initiation; it is not formylated, unlike prokaryotic fMet-tRNA

How is the binding of the initiator tRNA to the ribosome different in eukaryotes compared to prokaryotes?

In eukaryotes, tRNAiMet binds to the ribosome before associating with mRNA. In prokaryotes, the tRNA binds directly to the mRNA-ribosome complex at the start codon

Which complex binds to the mRNA first, and how?

The eIF4F complex binds first via eIF4E, which recognizes the 5' cap

What happens after both complexes bind to the mRNA?

The mRNA undergoes circularization, which promotes ribosome recycling

What mediates mRNA circularization in eukaryotes?

eIF4G links eIF4E (5' cap) to PABP (poly-A binding protein at 3' end)

What is the purpose of circularizing the mRNA?

It enhances ribosomal recycling for efficient translation re-initiation

How does the small ribosomal subunit find the start codon?

It scans from 5' to 3' using ATP and helicases until it finds the first AUG

Do eukaryotic mRNAs have a Shine-Dalgarno sequence?

No. Eukaryotes lack a Shine-Dalgarno sequence. The first AUG is identified by scanning

What happens when the correct AUG is recognized?

The codon-anticodon match triggers GTP hydrolysis by eIF2, causing conformational changes

What is the final step of initiation after AUG recognition?

The large (60S) ribosomal subunit joins, the initiator tRNA enters the P site, and all eIFs dissociate, forming the 80S initiation complex

Where does the amino acid attach on a tRNA molecule?

To the 3'OH of the ribose in the terminal adenine of the CCA acceptor arm, via an ester linkage

What is puromycin structurally similar to?

The adenine residue of the tRNA acceptor arm attached to an amino acid

How does puromycin enter the ribosome?

It mimics a charged tRNA and fits into the A site

What type of bond links the amino acid to puromycin?

An amide linkage (unlike the normal ester linkage in tRNA)

What happens when puromycin enters the A site?

Its nitrogen nucleophilically attacks the growing polypeptide chain, forming a peptide bond

Why does puromycin halt translation?

After translocation, its amide linkage prevents the next amino acid from attacking, stalling the ribosome and causing dissociation

Does puromycin affect prokaryotes, eukaryotes, or both?

Both

How does streptomycin inhibit translation?

It prevents correct binding of initiator tRNA in prokaryotes

What chemical property of streptomycin contributes to its action?

Its basic nature from many amino groups

What does the A fragment of diphtheria toxin do?

It ADP-ribosylates EF2, halting ribosome translocation in eukaryotes

What molecule does diphtheria toxin cleave to carry out its function?

NAD⁺, producing ADP-ribose and nicotinamide

What is diphthamide?

A modified histidine residue on EF2 targeted by diphtheria toxin

What is ricin composed of?

Two chains: A (catalytic) and B (uptake), connected by a disulfide bond

How does ricin A chain inhibit translation?

It removes an adenine from 28S rRNA, preventing elongation factor binding

What type of enzyme is ricin?

An N-gl