The basis of life part 2 - translation

Continuing from last week post on transcription of DNA into mRNA, we’re now onto translation, the conversion of mRNA into a protein.

Firstly, a ribosome binds to the start of the mRNA molecule and reads the nucleotide sequence and then synthesizes the correct protein from amino acids, which amino acid depends of the 3 letter nucleotide codon within the mRNA chain. Each amino acid is made of a carboxyl group (COOH) and an amino group (NH2) with a side chain which depicts the amino acid. The ribosome reads the mRNA from the amino-terminal to carboxyl-terminal direction (so the amino group starts the chain and the carboxyl group ends the chain). The ribosome reads each triplet codon of the mRNA in turn and lines up the complimentary anticodon on the aminoacyl-tRNA (transfer RNA with an amino acid covalently attached) which binds to the correct codon by a hydrogen bond. When another aminoacyl-tRNA hydrogen bonds to the adjacent codon the two amino acids on the tRNA form a peptide bond between them. This process is repeated for all the triplet codons on the mRNA, growing into a polypeptide chain.

As with transcription this also happens in 3 stages:-

1) Initiation – in this stage the mRNA-ribosome complex is formed. The first codon, AUG (its compatible amino acid being Methionine) is attached to the anticodon of the initiator tRNA (first aminoacyl-tRNA). As the codon AUG can also appear in the middle of the mRNA two different tRNA’s are required. For the start codon tRNAf met is used and this type is called the initiator tRNA. Whereas with AUG codons in the middle of the mRNA sequence tRNAm met is used. In bacteria the first amino acid of the protein is always N-formylmethionine, therefore the aminoacyl-tRNA is fMet-tRNAf met.

Ribosomes have 3 binding sites. The initation tRNA binds directly to the P site, of the ribosome, unlike all other aminoacyl-tRNA. 3 initiation factors catalyse this process; IF1, IF2 and IF3. Different processes require different orders of initiation factors so only a couple are know and the rest are still unclear.

2) Enlongation – As the other codons are read along the mRNA; EF-Tu (an elongation factor) and GTP catalyse the reaction (the second aminoacyl-tRNA to bind to the A site); as the aminoacyl-tRNA/EF-Tu/GTP complex is formed which reduces the activation energy aminoacyl-tRNA peptide bond together creating a polypeptide. During elongation the incoming aminoacyl-tRNA binds to site A (aminoacyl-tRNA binding site) in the ribosome. Once it has bonded it moves across to site P (peptideyl-tRNA binding site) where the tRNA is linked on to the end of the growing polypeptide chain by a peptide bond. The last binding site is site E (exit site) where tRNA is bound before its release from the ribosome and after fore filling its duty in translation. The elongation cycle consists of 3 steps: -

* Step 1 – aminoacyl-tRNA binds to the A site – from when the second codon binds to site A – the binding requires GTP and EF-Tu (elongation factor). The GTP and EF-Tu have bound on to the aminoacyl-tRNA for it to bind to site A in the ribosome. Once the aminoacyl-tRNA has been bonded to site A, the complex is broken down by the hydrolysis GTP into GDP which is bound to the EF-Tu. However, before the next aminoacyl-tRNA can be catalysed on to site A, the EF-Tu has to be regenerated.

* Step 2 – peptide bond formation – In the large ribosomal subunit lies peptidyl transferase which catalyses this stage of elongation. Firstly the carboxyl end of the amino acid is uncoupled from the tRNA; this happens in the P site. Then the carboxyl end of the amino acid undergoes a condensation reaction with the amino group of the aminoacyl-tRNA bound to site A, creating a peptide bond.

* Step 3 - translocation – this stage involves transolcase, a complex of elongation factors EF-G, binding to the ribosome with the GTP (EF-G/GTP complex binds to the ribosome). The tRNA, now deacylated, moves along the ribosome to the E site, and then the dipeptidyl-tRNA also moves across the ribosome from site A to P. The ribosome then moves across the mRNA so that the next codon is position in site A ready for the corresponding aminoacyl-tRNA.

3) Termination – There are 3 termination codons. Once one of the termination codon appear of the mRNA the polypeptide is transferred to a water molecule instead of an aminoacyl-tRNA, effectively cleaving the bond between the tRNA and the polypeptide chain in the P site. This is caused by the release factors, RF1 and RF2. The three possible termination codons are UAG, UAA and UGA however bacteria do not contain aminoacyl-tRNA complimentary to these stop codons. Instead of this a release factor binds instead. Also a third release factor is needed to assist either RF1 or RF2, this is known as RF3. RF1 or RF2 bind at or near the A site where as RF3 will bind elsewhere on the ribosome.

How protein synthesis can be targeted by antibacterial drugs

Antibacterial drugs only inhibit protein synthesis in prokaryotic cells and do not cause any damage to eukaryotic cells as there is a notable difference between the ribosomes in these different types of cell. Prokaryotic ribosomes contain two subunits, a small (30s) and a large (50s).

Different antibacterial drugs have different effects on protein synthesis. Different classes of antibacterial drugs will affect different stages in protein synthesis, some affect DNA being transcribed into mRNA whereas others can affect the translation process.

Chloramphenicol prevents protein synthesis as it inhibits mRNA from binding to the 50s subunit of the ribosome; stopping the mRNA from being translated into a protein.

Macroclides (eg erythromycin) and clindamycin affect protein synthesis by prohibiting the movement from site A to site P within the ribosomal mRNA complex. Therefore only 1 aminoacyl-tRNA (met) can bind with the ribosome and stopping protein synthesis.

Tetracycline inhibits the 30s subunit of the ribosomes blocks the aminoacyl-tRNA from joining the ribosome to begin with, stopping the Met-tRNAmet from bonding with the A site. Blocking the 30s subunit of the ribosome also stops the mRNA from binding to the ribosome to be translated, as mRNA needs to bind to both the 30s and 50s subunits. Therefore, synthesis cannot begin.

Aminoglycosides inhibits the specific binding of the mRNA to the 30s subunit of the ribosomes.

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