Translation- Part 2

This lecture doesn't have an outcomes slide, so I'm just going to expand on the summary as much as possible.

There are three potential translational frames in any mRNA; however, only one is used.

Translation of an mRNA can occur in one of three "reading frames." Let's say that we have an mRNA with bases 123456789. In the first reading frame, the first codon starts with base 1, so we have codons 123/456/789. In the second reading frame, the first codon starts with base 2, so we have codons 234/567. In the third reading frame, the first codon starts with base 3, so we have 345/678. There is no fourth reading frame because the end result is essentially the same as the first reading frame, minus the beginning codon. Although there are three different reading frames, only one will be used. The frame that will be used is usually one that produces a long chain without a stop codon.

Translation occurs in three steps: initiation, elongation and termination.

I will expand on these steps throughout the rest of this post...

Initiation of translation requires initiator Met-tRNAi in eukaryotes and fMet-tRNAi in prokaryotes.
When Met appears inside an open reading frame, Met-tRNA is used and this binds elongation co-factors.

As I may or may not have mentioned before, the codon that codes for methionine also codes for the start codon. Hence the initiating tRNA is always Met-tRNAi (the i stands for "initiator"). In prokaryotes, this initiating tRNA is slightly different- it's fMet-tRNAi, or N-formylmethionine tRNA, which is essentially Met-tRNAi bound to a formyl (aldehyde) group. The initiating tRNA is bound when the ribosome reaches a start codon. (This doesn't always happen- if I remember correctly sometimes the ribosome skips some start codons, resulting in "leaky scanning" and a different protein being produced. I'm not sure whether this happens in both eukaryotes and prokaryotes or only in eukaryotes, however.)

There are sequences on the mRNA to help guide the ribosome towards the correct start codon. In prokaryotes, the small subunit of the ribosome attaches to an initiation factor (IF), and then this complex binds to a special sequence called the Shine Dalgarno sequence, located upstream of the start codon. fMet-tRNAi then aligns with the start codon. The large subunit then binds, releasing IF. In eukaryotes, Met-tRNAi first binds to the small subunit, and then the loaded subunit scans along the mRNA to find the first AUG codon surrounded by a long consensus sequence known as the Kozac sequence. Initiation factors then detach, allowing the large subunit to bind.

Another important point to note is that the initiating Met-tRNAi is different to the Met-tRNA used in elongation. Met-tRNA has a slightly different shape, allowing it to bind more efficiently to elongation cofactors.

Elongation is a process using ATP to move the ribosome in the 5’ to 3’ direction, inserting tRNAs above codons and catalysing peptide bond formation between peptides.

During the process of elongation, the ribosome moves down the mRNA from 5' to 3', a codon at a time. At each position, an aminoacyl-tRNA enters the A site, the peptide in the P site joins onto the amino acid bound to the tRNA in the A site, and then the ribosome moves along. When the ribosome moves along, the tRNA originally in the A site goes into the P site, and the tRNA originally in the P site goes to the E site, where it leaves the ribosome. I've described this in more detail in an earlier post.

Termination occurs when the ribosome encounters a stop codon in the A-site, leading to a hydrolysis reaction, releasing the peptide from the final tRNA and causing dissociation of the ribosome from the mRNA. 

When the ribosome encounters a stop codon, release factors, rather than amino acids, bind. These release factors alter the activity of the peptidyl transferase (the enzyme that catalyses the addition of amino acids onto the chain). They also add water to the peptide, releasing it into the cytosol. The ribosome is then free to dissociate from the mRNA.