Gene Expression: Translation

Overview of Translation

Translation is the process by which the genetic information encoded in mRNA molecules is used to synthesise polypeptides. During translation, the mRNA's nucleotide sequence is decoded into a sequence of amino acids.

Translation of mRNA by a ribosome Image source: LadyofHats - Own work using:nih.govmiami.edumolecularassembler.comufl.eduphschool.comuic.eduyoutube.comyoutube.com, Public Domain, https://commons.wikimedia.org/w/index.php?curid=4889777

Nucleotides in the mRNA are read in sets of three, known as codons. Each codon corresponds to a specific amino acid or serves as a signal to start or stop protein synthesis. These codons are recognised by adaptor molecules called transfer RNAs (tRNAs).

Transfer RNAs carry specific amino acids to the ribosome, a large macromolecular complex where protein synthesis occurs. The ribosome catalyses the formation of peptide bonds between adjacent amino acids, linking them together to form a growing polypeptide chain.

Transfer RNAs

A tRNA is a relatively short RNA molecule, typically about 80 nucleotides long, that recognises a specific codon in the mRNA and matches it with an appropriate amino acid.

The tRNA molecule folds into a complex structure due to the formation of four double-helical segments held together by intramolecular hydrogen bonds. When drawn in one plane, the tRNA resembles a cloverleaf, while its 3D structure is L-shaped.

(a) The two-dimensional tRNA structure resembles a cloverleaf (b) The three-dimensional tRNA structure is L-shaped Image modified from: CNX OpenStax - https://cnx.org/contents/[email protected], CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=53713134

At the 3' end of the L-shaped molecule, an amino acid is covalently attached to the tRNA through an ester bond, forming an aminoacyl-tRNA molecule. The specific amino acid attached to the tRNA corresponds to the codon recognised by the tRNA during translation. The loop at the tip of the L-shaped tRNA contains the anticodon, a sequence of three nucleotides complementary to a specific codon in the mRNA.

Although there are 61 codons coding for amino acids, the number of distinct tRNA molecules does not necessarily match this count. Instead, the actual number of tRNAs varies among species, with some bacteria having as few as 31 tRNAs and humans possessing 48 tRNAs.

Normal and wobble base-pairing

The reason for this discrepancy is that the structure of some tRNA molecules allows them to bend the rules of complementary base-pairing in the third position of a codon – a phenomenon known as wobble base pairing. In those cases, accurate codon-anticodon recognition is required only for the first two nucleotide pairs while the third nucleotide pair can be mismatched. For instance, in bacteria, a guanine nucleotide in the third position of a codon can pair with either cytosine or uracil. The wobble base-pairing allows one tRNA to recognise several codons despite differences in their nucleotide sequences.

Ribosome Structure and Function

Ribosomes are large molecular machines that catalyse protein synthesis. They are composed of ribosomal RNAs (rRNAs) associated with ribosomal proteins, and while the exact number of rRNAs and proteins may vary across different organisms, the general organisation of the ribosome remains rather conservative. A ribosome comprises a large and a small subunit, that come together around the mRNA and separate after the protein synthesis is complete so that they can be re-used.

The large ribosomal subunit (red) and small ribosomal subunit (blue). For the large subunit, the rRNAs are shown in dark red and orange-red, and the ribosomal proteins are shown in pink. For the small subunit, the rRNA is shown in dark blue and the ribosomal proteins in light blue). Image source: Vossman - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=6865434

The ribosome has three binding sites for tRNAs, namely the A (aminoacyl), P (peptidyl), and E (exit) sites. Transfer RNAs move sequentially from one site to another during translation. The A site is responsible for binding the tRNA carrying the next amino acid to be added to the growing polypeptide chain. The P site holds the tRNA, which carries the growing polypeptide chain. The E site facilitates the exit of tRNA molecules that have already participated in translation.

The tRNA binding sites in the ribosome: A (aminoacyl), P (peptidyl), and E (exit) sites Image source: Untitled image. "Ribosome". Accessed March 18, 2024. https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-polypeptides/a/the-stages-of-translation

The catalytic activity in the peptide bond formation is performed by the rRNA component of the ribosome. This enzymatic activity makes ribosomes ribozymes—catalytically active RNA molecules.

Unlike prokaryotic ribosomes, in eukaryotes, some ribosomes synthesise proteins when docked on the internal membranous structures of the cell (i.e. on the rough endoplasmic reticulum). In that case, a produced protein is compartmentalised to this membranous structure, can be inserted into cell membranes, or destined for secretion.

The Process of Translation

Translation involves three major stages: initiation, elongation and termination.

Initiation of translation

At the initiation stage, the small subunit of the ribosome first binds to a specialised initiator tRNA. This initiator tRNA carries the amino acid methionine (or formyl-methionine in bacteria). The initiator tRNA has a unique ability to bind directly to the P site of the ribosome without initially passing through the A site.

In prokaryotes, translation initiation is guided by the presence of a sequence known as the Shine-Dalgarno sequence, located upstream of the start codon on the mRNA. This sequence helps position the initiator tRNA precisely at the start codon. In eukaryotes, the small ribosomal subunit, with the initiator tRNA already on board, associates with the 5' cap of the mRNA first. The ribosome then scans along the mRNA until it encounters the start codon.

Initiation of translation in prokaryotes Image source: Untitled image. "Initiation of translation". Accessed March 18, 2024. https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-polypeptides/a/the-stages-of-translation

Once the initiator tRNA is correctly positioned at the start codon, the large ribosomal subunit joins the complex, forming a complete ribosome. The A site is now ready to receive the next aminoacyl-tRNA and the translation can start.

Translation initiation in both prokaryotes and eukaryotes requires the assistance of specific protein factors. The process also requires the expense of energy in the form of guanosine triphosphate (GTP).

Elongation

After the translation initiation is complete, the elongation cycle begins, during which amino acids are added to the growing polypeptide chain. This process proceeds from the amino group end (N-terminus) towards the carboxyl end (C-terminus) of the polypeptide.

The elongation cycle Image source: Untitled image. "The elongation cycle". Accessed March 19, 2024. https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-polypeptides/a/the-stages-of-translation

The elongation cycle involves several steps assisted by a set of specific protein factors:

1. Codon recognition. In this step, an aminoacyl-tRNA enters the A site and binds the codon if codon-anticodon recognition occurs properly. To ensure accurate tRNA selection, a molecule of GTP is expended.

2. Formation of the peptide bond. The growing polypeptide chain, held by the peptidyl-tRNA located in the P site, is transferred to the amino acid attached to the aminoacyl-tRNA in the A site.

3. Translocation. In this step, the peptidyl-tRNA moves from the A site to the P site. Simultaneously, the discharged tRNA in the P site moves to the E site and vacates the ribosome. The mRNA, bound to the tRNA, also moves, bringing the next codon into the A site for the next cycle of elongation. The translocation step requires the hydrolysis of one GTP molecule.

These steps repeat iteratively until a stop codon is encountered on the mRNA, signalling the termination of translation.

Termination

Translation terminates when the ribosome encounters a stop codon. The stop codons are recognised by no tRNA but can be bound by a protein called release factor which mimics the tRNA shape to occupy the A site. The release factors catalyse the hydrolytic reaction which cleaves the polypeptide from its tRNA in the P site thus allowing the polypeptide to be released from the ribosome.

Summary

The video below summarises the translation process and provides additional details on protein factors: