Ribosome & Protein Translation: Eukaryotic

Eukaryotic Ribosome

• the eukaryotic ribosome is structurally similar to the prokaryotic complex, but larger

• assembly of the eukaryotic ribosome requires the coordination of several events across diff cellular compartments

Eukaryotic Translation

• key difference: transcription occurs in the nucleus and translation occurs in the cytosol (processes are uncoupled in eukaryotes)

• rRNAs are processed in the nucleolus, while small ribosomal proteins are synthesized in the cytosol before being imported and assembled into pre-ribosomal particles (pre-40s and pre-60s)in the nucleolus with the help of assembly factors

• the pre-40s and pre-60s subunits are then exported to the cytosol, and assembly factors are recycled

• eukaryotic translation is similar to the prokaryotic process but has major distinctions especially in the initiation process

Factors binding to mRNA: PABP

• important factors bind to mRNA in the cytosol, including the 3’ poly(A)-binding protein (PABP)

• PABP interacts w/ eukaryotic initiation factors (eIFs) bound to the 5’ cap

  • this interaction promotes circularization of the mRNA, enhancing both translation efficiency and mRNA stability

43S Pre-Initiation Complex

• eukaryotic initiation involves multiple eukaryotic initiation factors (eIFs) that assemble with the 40S ribosomal subunit to form the 43S pre-initiation complex

• the 43S pre-initiation complex contains the Met-tRNA_i (unmodified Met but dedicated initiation tRNA) in the P site delivered by eIF2-GTP

• similar to prokaryotic initiation, eIFs block the A site and prevent premature association with the 60S large subunit

  • also like in prokaryotic initiation, there are 2 tRNAs that recognize Met (AUG) codons, one is used during elongation and one during initiation

    • these tRNAs are activated w/ unmodified Met

mRNA Scanning & Start Codon Recognition

• with the assistance of additional eIFs, the mRNA is recruited

• the 43S pre-initiation complex after mRNA has been recruited (48S initiation complex) scans along the mRNA to identify the AUG start codon (the start codon can be put in the A site)

  • the scanning process requires helicase activity and ATP hydrolysis to unwind 2º structures in the mRNA

• only after GTP hydrolysis and dissociation of initiation factors is the 60S subunit recruited to form the functional 80S ribosome

  • hydrolysis of eIF2-associated GTP occurs upon start codon recognition

Kozak Sequence

• Kozak sequence is a conserved motif and a consensus sequence that surrounds the AUG start codon in eukaryotic mRNAs

• it helps the scanning ribosome recognize the correct initiation site

• in contrast, the Shine-Dalgarno sequence in prokaryotes is located upstream of the start codon and base-pairs with 16S rRNA to place AUG on the P site

• the Kozak sequence typically contains a purine (A/G) in position -3 and a G in +4

Start Codon Recognition: Open → Closed Complex Transition

• during scanning, in the open conformation, eIF1 prevents the association of eIF5

• recognition of Kozak sequence elements (position -3 and +4) stabilizes multiple interactions with initiation factors and rRNA, promoting formation of the closed complex

• closed complex: Met-tRNA_i is properly positioned in the P site

  • eIF1 is evicted

  • eIF5 (GTPase activating protein) can now associate and promote GTP hydrolysis by eIF2

    • eIF5 is brought into proximity with eIF2-GTP

• GTP hydrolysis signals to ribosomes that the correct AUG is placed in the P site and to proceed with the last stage of initiation

Accuracy of Translation Elongation

• translation has no true proofreading, so why isn’t it more error-prone?

• because timing is everything!

• GTP hydrolysis be EF-Tu (prokaryotes)/eEF1A (eukaryotes) acts as a kinetic checkpoint linking translation speed to accuracy

  • role: delivers aminoacyl-tRNAs to the A site

  • GTP hydrolysis only occurs if codon-anticodon pairing is correct

Prokaryotic Accommodation

• accommodation is tightly linked to translation speed & accuracy

• it consists of a conformational change that repositions the tRNA within the A site, allowing peptide bond formation to occur

• accommodation only takes place after GTP hydrolysis and dissociation of elongation factor (Ef-Tu or eEF1A) in its GDP-bound form

Prokaryotic Accommodation: Mech

• upon initial binding, the incoming AA-tRNA is not positioned in a configuration that permits peptide bond formation (the 2 AA’s are far away from each other)

• incorrect tRNAs typically dissociate from the A site before GTP hydrolysis occurs, ensuring that only correctly paired AA tRNAs remain associated prior to accommodation

• following GTP hydrolysis, Ef-TU (GDP) is released, allowing accommodation to proceed

• once the incoming AA-tRNA is fully accommodated and correctly positioned, peptide bond formation can occur

GTP Hydrolysis

• GTP binds non covalently to translation factors

• upon GTP hydrolysis (GDP + Pi), the translation factor undergoes a conformational change

• each GTP-binding TF has a slow intrinsic GTPase activity

  • GTP hydrolysis is stimulated by a GAP (GTPase-activating protein)

TF Conformational Change

• the configuration of elongation factors is different when it is associated w/ GDP vs. an analog of GDP

• when EF-Tu elongation factor is bound to GDP, there’s high affinity to charged tRNA

GTP Hydrolysis Continued

• GTP hydrolysis by Ef-Tu is stimulated by the 50S ribosomal subunit acting as a GAP (GTPase activating protein)

• this timing ensures hydrolysis occurs only for correctly paired tRNAs

  • Correct codon–anticodon pairing stabilizes the tRNA long enough for GTP hydrolysis

  • Incorrect tRNAs dissociate before GTP hydrolysis

• GDP to GTP exchange is then mediated by EF-Ts acting as a GEF (GTP exchange factor)

  • EF-Ts promote dissociation of GFP so that new GTP can associate and elongation factors can be recycled

  • Ef-Tu-GTP is regenerated

Antibiotics & Translation Inhibition

• several small molecules inhibit translation

• because many of these molecules inhibit prokaryotic but not eukaryotic ribosomes, they can be used as antibiotics

• e.g. puromycin prematurely terminates translation by forming a peptide bond to release the elongated polypeptide, releasing it from the tRNA

• Cycloheximide inhibits eukaryotic ribosomes and is often used to measure protein turnover (in absence of translation)