July 11, 2001 — Genetic code

Five rules:

  1. All 64 codons have meaning (3 are stop codons, the rest encode aa.)
  2. Code is unambiguous: each codon only codes for one aa.
  3. Code is degenerate: a lot of aa have more than one codon
  4. Codons representing the same aa are often similar in sequence

    5. E.g. Gly is GGX, where X can be any one of the 4 bases. This provides an advantage for proteins because there is a greater chance a mutation will not change the aa in the protein.

  5. Code is universal: prokaryotes and eukaryotes use essentially the same code.

E.g. AUG is methionine in both. Therefore you can use bacteria as factories for eukaryotic proteins.

There are a few exceptions usually dealing with the stop and start codons.

E.g. In mycoplasma UGA-Trp instead of a stop codon

There are also changes in mitochondria codons, but these do not have a strong effect, since mitochondria only code10 proteins.

Wobble hypothesis — pairing between codons and anticodons is relaxed in third position of codon and first position of anticodon, because tRNA is flexible.

Can have G-U base pairs

Inosine — looks like adenosine but has double bonded O instead of an amino group, therefore can pair with U, G, and A (Fig. 7.6)

"Noncononical base pairing"

Advantages of relaxed base pairing?

  1. You have a weaker H-bond in the third position. This base pair can break up faster, therefore you get a quicker release in translation.
  2. You need fewer tRNA’s, instead of having individual ones for all codons.

Synthetases — enzyme that charges aa on tRNA

There are 20 synthetases, one for every aa, therefore there are ones that recognize more than one tRNA.

Fig. 7.1 UGG-Trp has one synthetase

But, Leu has 6 codons, but only one synthetase

Charging the tRNA is a critical step for translation accuracy- 2 step reaction

  1. linkage between aa and ATP ----> aminoacyl AMP (has a phosphate group)
  2. aa transferred to tRNA. The amino acid now no longer has any influence over translation, the only thing that determines what aa is added to the growing polypeptide is the codon and anticodon interaction.

Show this through an experiment where you take cysteine and remove the sulfur to form an alanine. The tRNA still recognizes the cysteine codon, but all the cysteines are replaced by alanines in the protein.

To assure correct charging, there are mechanisms for proofreading: (Fig 7.13)

  1. kinetic proofreading — involved in binding of the correct tRNA to synthetase

    2. A perfect tRNA will associate quickly and dissociate slowly (and the incorrect tRNA will associate slowly and dissociate quickly.) This increases the chance that the right tRNA is amino acylated (with a covalent bond). This is a time dependent mechanism.

  2. chemical proofreading — acts at two stages to make sure you get the correct aa.

Works by making and breaking a bond.

1st chance: occurs after activation and before charging. The phosphate will be cleaved and the aa bumped out of the synthetase.

2nd chance: occurs after charging reaction. The bond will be hydrolyzed and the aa expelled.

Both of these proofreading mechanisms depend greatly on how everything fits together, their 3D interactions.

Enigma: synthetase sequences are highly diverse at the molecular level, despite their similar functions.

Two types of sythetases:

Class I and Class II (Fig. 7.11/12) — Each approach tRNA from a different side because they recognize different surfaces of the molecule.

Examples of things that go wrong and the ways to fix a stop codon

Nonsense mutation — a base change that turns a codon for an aa into a stop codon which results in premature truncation of the protein.

Can avoid its effect through a second mutation in the tRNA to form a "nonsense tRNA suppressor" (Fig. 7.16): the tRNA anticodon is mutated so that it now recognizes a stop codon (there are normally no tRNA’s that recognize stop codons. Stop codons are recognized by release factors).

The tRNA base pairs and inserts an aa instead of a truncation of the ORF.

The "nonsense tRNA suppressor" will be competing with the release factors normally in the cell.

Missense suppressor tRNA (Fig. 7.17)

An anticodon is mutated so that it recognizes a different triplet.

A very rare occurrence, but fairly deleterious.

Frameshifting

You have an ORF, and the ribosome is marching along the mRNA. The ribosome may frameshift, or do translational jumping where its skips a codon.

Tend to occur at sequences where there is secondary structure (similar to having a hairpin during transcription, which causes a ribosome to pause)

Example where frameshifting is a normal regulatory mechanism, and where something’s abundance affects its own translation:

Release factor II (RFII) is responsible for binding to A-site and recognizes 2 specific stop codons.

In the RFII coding sequence, there is a STOP codon in the middle of the ORF (as well one at the end, in the n+1 reading frame)

The ribosome moves along the ORF until it reaches the first stop codon:

If there is enough RFII, it binds to stop codon and termination results. You do not make a full length RFII.

If there is not enough/ or no RFII, ribosome pauses because nothing is put into A site. When it pauses, it will "hiccup" and change frames, by advancing one base. Translation now occurs until the second stop codon is reached and a full length RFII produced.

This only works in bacteria because it has two release factors, while eukaryotes only have one release factor.