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Everything here describes translation in prokaryotes, specifically Escherichia coli . Eukaryotes use a larger ribosome (80S, made of the 40S and 60S subunits), locate the start of the message from its 5 ′ end instead of a Shine-Dalgarno sequence, begin with unformylated methionine, and need more initiation factors. Where no organism is named, assume prokaryote.

Before we start

The components of translation and its six steps, one per slide: recognizing the message, initiation, reading the codon, the peptide bond, translocation and termination. Plus the molecule the process produces.

In this document

How the ribosome turns a sequence of bases into a chain of amino acids. The whole process in prokaryotes, with E. coli as the model. From message to protein

Central dogma

Most amino acids have more than one codon. Leucine has six; tryptophan has just one. Codons for the same amino acid usually differ in the third base, so many mutations at that position leave the protein unchanged. The code is nearly identical across all organisms, with a few exceptions, several of them in mitochondria.

Redundancy

With two bases per codon there would be only 16 combinations, too few for 20 amino acids. Three is the minimum that covers them all, with combinations to spare.

Why three bases

Four bases in groups of three give 64 possible combinations. Of these, 61 code for amino acids and three (UAA, UAG and UGA) mark the end of the protein. The AUG codon does two jobs: it codes for methionine and signals where reading begins.

The genetic code

Messenger RNA (mRNA) writes with four bases, and proteins are built from twenty amino acids. The cell reads mRNA in groups of three bases called codons . 64T ranslating between two alphabets codons for 20 amino acids

The problem

The third base of the codon pairs less rigidly than the first two, so a single anticodon can recognize several codons that differ only at that position. That is how E. coli reads its 61 codons with fewer than 50 types of tRNA.

Wobble

There is one aminoacyl-tRNA synthetase for each amino acid. Each recognizes its amino acid and the tRNAs that match it, and joins them using ATP. Many have an editing site that hydrolyzes the amino acid if it is the wrong one. The ribosome only checks the pairing between codon and anticodon: if a tRNA arrives loaded with the wrong amino acid, that amino acid goes into the protein. The code is actually translated at the synthetases.

The synthetases

A tRNA is 73 to 93 nucleotides long and folds into an L shape. The anticodon sits in a loop at one end; the amino acid attaches to the 3 ′ end, which always finishes in the sequence CCA. The two ends lie about 7 nanometers apart.

The structure

Each transfer RNA (tRNA) carries an anticodon at one end that pairs with the codon, and at the other end the amino acid that matches it. Aminoacyl-tRNA synthetase does the loading. T ransfer RNAThe piece that translates

The components

The S measures sedimentation rate in an ultracentrifuge, which depends on mass and also on shape. That’s why the values don’t add up: a 30S subunit and a 50S subunit form a 70S ribosome.

The svedberg unit

The A site (aminoacyl) receives each loaded tRNA as it arrives. The P site (peptidyl) holds the tRNA carrying the growing chain. The E site (exit) holds the empty tRNA before it leaves the ribosome. Every tRNA passes through all three, in that order.

The three sites

The 30S subunit contains the 16S ribosomal RNA and 21 proteins. The 50S contains the 23S and 5S ribosomal RNAs and about 33 proteins. Two thirds of the ribosome’s mass is RNA.

The composition

The 30S and 50S subunits assemble into the 70S ribosome. Inside are three sites that tRNAs pass through: A, P and E . 70S ribosomeThe ribosome

The components

In 1974, John Shine and Lynn Dalgarno sequenced the 3 ′ end of E. coli 16S ribosomal RNA and proposed that it paired with a sequence found in mRNAs just before the start codon.

Where the name comes from

An mRNA contains many AUGs, and most of them code for methionine inside the protein. The Shine-Dalgarno sequence marks which one is the start. Bacterial mRNAs often carry several genes in a row, each with its own Shine-Dalgarno sequence, so one message can have several start points. In bacteria, the start can also be GUG or UUG.

S needed

The Shine-Dalgarno sequence is purine-rich, with a consensus close to AGGAGG, and sits five to ten nucleotides before the start codon. It is complementary to the 3 ′ end of the 16S ribosomal RNA. Pairing between the two places the AUG right at the position of the P site.

The mechanism

The Shine-Dalgarno sequence on the mRNA pairs with the 16S ribosomal RNA of the 30S subunit. That pairing puts the AUG start codon in place. 30S subunitFinding the start

Step 1 of 6

Human neutrophils have receptors that detect formylmethionine peptides and read them as a sign of bacterial infection. Mitochondria also start their proteins with formylmethionine, so mitochondrial damage can release those peptides and set off an inflammatory response with no bacteria present.

The connection

It is a methionine with a formyl group added, attached once the amino acid is already bound to the initiator tRNA. That group sets apart the tRNA that starts reading from the one that places methionines inside the protein. After synthesis, a deformylase removes the formyl group, and in many proteins an aminopeptidase also removes the methionine.

Formylmethionine

Three initiation factors set up the assembly. IF3 keeps the 50S subunit from joining too early, IF1 occupies the A site, and IF2, bound to GTP, positions the initiator tRNA. When the 50S subunit arrives, IF2 hydrolyzes its GTP and all three factors leave. The initiator tRNA is the only one that enters the P site directly; every other tRNA arrives through the A site.

The mechanism

A special tRNA carrying formylmethionine settles onto the AUG. Then the 50S subunit joins and the complete 70S ribosome forms. Initiator tRNAInitiation

Step 2 of 6

Several antibiotics act on this step. Tetracyclines keep tRNA from entering the A site. Aminoglycosides such as streptomycin bind the decoding center and cause misreading. They work through selective toxicity: the bacterial ribosome differs from the human cytoplasmic ribosome, but mitochondria keep ribosomes similar to bacterial ones, which accounts for part of the hearing damage aminoglycosides can cause.

The connection

The ribosome checks the pairing twice: before EF-Tu hydrolyzes its GTP and again afterward. A wrong tRNA gets two chances to fall off. The result is roughly one wrong amino acid per 10⁴, at about 20 amino acids per second.

The double check

EF-Tu, bound to GTP, carries the loaded tRNA. In the decoding center of the 30S subunit, two bases of the 16S ribosomal RNA (A1492 and A1493) inspect the geometry of the first two codon-anticodon pairs. A correct match changes the ribosome’s shape, EF-Tu hydrolyzes its GTP and leaves, and the tRNA settles into the A site.

The mechanism

EF-Tu delivers each loaded tRNA to the A site . If the anticodon pairs with the codon, the tRNA stays; if it doesn’t, the tRNA leaves. EF-T uReading the codon

Step 3 of 6

Chloramphenicol binds the peptidyl transferase center and blocks bond formation. It also inhibits protein synthesis in human mitochondria, which contributes to the bone marrow suppression that limits its clinical use.

The connection

The catalyst for the peptide bond is the 23S ribosomal RNA. In 2000, Nissen and colleagues solved the structure of the large subunit and showed that no protein side chains come within about 18 Å of the bond being formed. The ribosome is a ribozyme, a finding that supports the hypothesis that RNA catalyzed reactions before proteins existed.

An enzyme made of rna

The amino group of the amino acid in the A site attacks the ester bond linking the chain to the tRNA in the P site. The chain becomes attached to the new amino acid, grows by one unit, and now hangs from the A-site tRNA. The P-site tRNA is left empty.

The mechanism

In the 50S subunit, the peptidyl transferase center forms the peptide bond . The chain moves from the tRNA in the P site onto the amino acid in the A site. Peptidyl transferase centerJoining the amino acids

Step 4 of 6

At about 20 amino acids per second, E. coli makes a 300-amino-acid protein in roughly 15 seconds. EF-Tu, which takes part in every cycle, is one of the most abundant proteins in the cell. The antibiotic fusidic acid blocks this step by trapping EF-G on the ribosome.

The speed

Each amino acid added costs the equivalent of four high-energy phosphate bonds: two when the synthetase loads the tRNA, one in EF-Tu’s GTP and one in EF-G’s GTP. Protein synthesis is one of the most energy-hungry processes in a growing bacterium.

The cost

After the peptide bond forms, the subunits rotate slightly relative to each other and the tRNAs sit in intermediate positions between sites. EF-G, bound to GTP, docks on the ribosome, and GTP hydrolysis drives the mRNA and both tRNAs forward by one codon. The A site is left free for the next tRNA.

The mechanism

EF-G uses energy from GTP to move the ribosome exactly three bases . The tRNA in the P site shifts to E and leaves; the one in the A site shifts to P. EF-GMoving one codon

Step 5 of 6

A single base change can turn an amino acid codon into a stop codon. The ribosome halts too early and the protein comes out incomplete. Some cases of cystic fibrosis and Duchenne muscular dystrophy are caused by this type of mutation.

Nonsense mutations

RF3, bound to GTP, removes the release factor. Then ribosome recycling factor and EF-G split the subunits apart, and IF3 binds the 30S to keep them separate until the next round of initiation.

Recycling

RF1 recognizes the UAA and UAG codons; RF2 recognizes UAA and UGA. The factor occupies the A site and reaches from the decoding center to the peptidyl transferase center, where a three-amino-acid motif (GGQ) positions a water molecule. That water hydrolyzes the bond holding the chain to the P-site tRNA, and the protein is released.

The mechanism

The UAA, UAG and UGA codons have no tRNA. When one reaches the A site, a release factor enters, and the peptidyl transferase center lets the chain go. Release factorsT ermination

Step 6 of 6

The amino acid sequence is the primary structure. To work, the chain has to fold into its three-dimensional structure, and many proteins also need modifications or have to pair up with other chains.

S left

One ribosome doesn’t have to finish before another starts: as soon as the first moves away from the start, the next can bind the same Shine-Dalgarno sequence. Combined with the coupled transcription from folder 3, a single mRNA can be synthesized at one end while several ribosomes translate it at the same time.

The polysome

The chain passes through the 50S subunit via a tunnel about 100 Å long that holds 30 to 40 amino acids. Folding can begin inside it, and on the way out the chain is met by a chaperone called trigger factor. Macrolides such as erythromycin bind the tunnel and block the chain’s passage.

The exit tunnel

A chain of amino acids

The result

The chain exits through a tunnel in the 50S subunit and begins to fold. Several ribosomes read the same mRNA at once: a polysome .

- Alberts, B., Heald, R., Johnson, A., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2022). Molecular biology of the cell (7th ed.). W. W. Norton & Company. - Nelson, D. L., Cox, M. M., & Hoskins, A. A. (2021). Lehninger principles of biochemistry (8th ed.). W. H. Freeman. - Nissen, P., Hansen, J., Ban, N., Moore, P. B., & Steitz, T. A. (2000). The structural basis of ribosome activity in peptide bond synthesis. Science, 289(5481), 920–930. https://doi.org/10.1126/science.289.5481.920 - Rodnina, M. V. (2018). Translation in prokaryotes. Cold Spring Harbor Perspectives in Biology, 10(9), a032664. https://doi.org/10.1101/cshperspect.a032664 - Shine, J., & Dalgarno, L. (1974). The 3 ′-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proceedings of the National Academy of Sciences of the United States of America, 71(4), 1342–1346. https://doi.org/10.1073/pnas.71.4.1342

References

The interactive Central Dogma minigame is at aequorea.net, alongside the rest of the series’ downloadable resources. The full video walks through every one of these steps with 3D animation.

Keep studying

The information was born in DNA, passed through RNA and ended in a protein, in the direction Crick proposed in 1958. Folder 5 covers the exception: viruses that can turn RNA into DNA.

S next

The whole process, animated and explained in full, in the Central Dogma video. The cycle iscomplete