Protein Synthesis

Describe eukaryotic translation

- Prokaryotes, gene expression is controlled at level of transcription; do we express the RNA or not
- Eukaryotes have 2 levels: 1 in making RNA and 2 in making protein
- Overall rate of expression in cell can be controlled
- Decision to proceed is controlled by phosphorylation/dephosphorylation of initiation factors
- Eukaryotic elongation factor eEF1A is similar to prokaryotic EF1A (EF-TU)
o eEF1b is similar to GEF EF-Ts
- Initiation of protein synthesis is much more complex and requires a large number of eIFs, with some serving as
scaffolds (eIF3 and eIF4G)
o Eukaryote start aa is methionine, pro is formyl-methionine
- Pre-initiation complex forms, which includes initiation factors, small ribosomal subunit, and loaded initiatior
tRNA (met-tRNA)
o This binds to a separate complex that includes mRNA, IFs that interact with the 5’ methylguanosine cap
and 3’polyA tail
- After initiation complex assembles, it translocates along the mRNA called scanning until AUG is reached – this is
facilitated by eIF4A
o After initiation codon is recognized, there is hydrolysis of GTP and release of initiation factors, as the
large ribosomal subunit joins the complex and elongation commences
o Some eukaryotic mRNAs have an internal ribosome entry site (IRES) far from the 5’ capped end, at which
initiation may occur without the scanning process (can be used to make artificial polycistronic eukaryotic
mRNA)

Identify polyribosomes

- Cluster of ribosomes reading the same mRNA simultaneously producing the same protein at high rates

Explain function of miRNAs

- Broader target spectrum than siRNA, translated from a completely different gene
- If miRNA matches sequence of target RNA, it will target it for degradation and does not need to be perfectly
complementary (if not perfect, inhibits translation)

Describe possible mutations influencing translation

- Nonsense: premature stop codon UAA UGA or UAG

- Missense: point change in nucleotide sequence, causing 1 amino acid to be substituted for another
- Frameshift: addition or deletion of bp, changing reading frame
- Synonymous: mutation does not change amino acid
- Non-synonymous: mutation does change amino acid ; missense

Identify the steps in nonsense mediated decay and the role of the exon junction complex

- Nonsense mediated decay is a way that cells can degrade the nonsense mRNA before it gets translated, it takes
place in mRNAs with introns and it usually requires the mutation upstream of the exon
- Normally, Exon junction complex where 2 exons are joined together
o Pioneer translation happens (nucleus, probably cytoplasm)
o Form ribosome structure doing a very brief burst of translation, once it reaches the EJC, it changes a few
things (EJC remodeling), which allows the complex to become stabilized and it goes on normally
- In transcripts with nonsense codons
o Splicing occurs, but when Pioneer translation occurs, it never reaches EJC so no remodelling of EJC,
which produces surveillance complex, which causes degradation of the mRNA
 - There are also non-stop delays: You don't want the mRNA to be constantly translated, so if you have a situation
with no stop codon, a similar mechanism degrades the aberrant mRNA

Explain the possibilities of experimentally manipulating codons/amino acids to make novel proteins

- Scientists are synthesizing new amino acids to create new proteins to have new functions
- Freeing up codons to produce them by eliminating redundant codons