Biology › Nucleic acids, genomes and protein synthesis › Translation: turning a message into a working protein
Translation: turning a message into a working protein
A ribosome grips two codons at a time, holds two tRNAs alongside them, makes one peptide bond and shuffles on. Repeat a few hundred times and a polypeptide falls off the end. The folding that goes on alongside it, and the trimming and the sugars added in the Golgi afterwards, are what turn a chain into a protein that works.
Before this The genetic code and transcription · Protein structure and folding · The Golgi apparatus and secretion · ATP as an energy currency
Before you start
tRNA carries the codon to the ribosome, where it is matched against the mRNA. Every word of that is in the right place but one, and it is the one carrying the mechanism. The codon is on the mRNA; the tRNA carries the anticodon, which is the complementary triplet. The mRNA codon CCU is read by a tRNA whose anticodon is GGA, and that tRNA is the one carrying proline. Get the two words the wrong way round in an exam answer and the sentence describes a mechanism that could not work, because two identical triplets would have nothing to pair with.
What you should be able to do
- Describe the structure of a ribosome and say what its two binding sites hold.
- Explain the relationship between a codon and an anticodon, and say which molecule carries which.
- Describe initiation, elongation and termination in order.
- Explain where the energy for protein synthesis comes from and roughly how much is needed.
- Describe what happens to a polypeptide after it leaves the ribosome.
- Work from a given DNA sequence to the amino acid sequence it specifies.
The machine: a ribosome and two sites
A ribosome is built from ribosomal RNA and proteins, in two subunits of unequal size that clamp around the mRNA. Eukaryotic ribosomes are 80S; those in prokaryotes, mitochondria and chloroplasts are the smaller 70S, which is a fact worth holding on to because several antibiotics work by binding to 70S ribosomes and leaving the 80S ribosomes in your cytoplasm untouched. Note where that leaves your mitochondria, whose ribosomes are 70S: those drugs bind there too, which is where a good deal of their toxicity comes from.
The two binding sites are usually called the P site and the A site. The P site holds the tRNA carrying the growing polypeptide chain; the A site accepts the next tRNA, whose anticodon must match the codon underneath it. Because the ribosome moves along the mRNA in the 5′ to 3′ direction, the A site is always the one further along the message.
- Ribosome
- An organelle of ribosomal RNA and protein, in two subunits, which holds mRNA and tRNA in position while a polypeptide is assembled.
- Codon
- A triplet of bases on mRNA specifying one amino acid or a stop signal.
- Anticodon
- A triplet of bases on tRNA, complementary to a codon on mRNA.
tRNA: the adaptor that makes the code physical
Nothing about a triplet of bases resembles an amino acid, so something has to connect the two. That something is tRNA. Each tRNA molecule is about 80 nucleotides long, folded by hydrogen bonding between complementary parts of its own sequence into a clover-leaf shape with an anticodon at one end and a site for one specific amino acid at the other.
Loading a tRNA with its amino acid is done by a set of enzymes, one for each amino acid, and it costs ATP. The pairing is specific: a tRNA with anticodon GGA is loaded with proline and nothing else. That specificity is where the genetic code physically lives — the code is not a property of the ribosome, which simply matches triplets, but of which amino acid gets attached to which tRNA.
The pairing at the ribosome then follows the ordinary rules. Anticodon and codon are held together by hydrogen bonds between complementary bases, with adenine pairing to uracil and guanine to cytosine. Those bonds are weak, which is what lets the tRNA leave again once its amino acid has been handed over.
Initiation, elongation, termination
Translation is easiest to write about as three stages, and mark schemes generally follow that structure.
Initiation. The mRNA binds to the small subunit of a ribosome. The ribosome moves along to the start codon, AUG, and the tRNA with the complementary anticodon UAC — carrying methionine — binds in the P site. The large subunit joins, and the ribosome is assembled around the message.
Elongation. A second tRNA, whose anticodon matches the next codon, binds in the A site. The two amino acids are now adjacent, and the ribosome catalyses the formation of a peptide bond between them by condensation, releasing water. The first tRNA, now empty, leaves. The ribosome moves along exactly one codon, which shifts the remaining tRNA into the P site and leaves the A site free for the next one. That cycle repeats, once per amino acid.
Termination. When a stop codon — UAA, UAG or UGA — reaches the A site, no tRNA has a matching anticodon. Instead a release factor binds, the completed polypeptide is freed, and the ribosome separates into its subunits, ready to start again. Notice that the stop codons specify no amino acid at all, which is why a message of 300 codons ending in a stop yields a polypeptide of 299 amino acids.
| Stage | What happens | What to name in an answer |
|---|---|---|
| Initiation | mRNA binds the small subunit; the start codon AUG is found | AUG, methionine, the P site |
| Elongation | tRNAs arrive in the A site; peptide bonds form; the ribosome shifts one codon | Codon-anticodon pairing, condensation, peptide bond |
| Termination | A stop codon reaches the A site and no tRNA matches | UAA, UAG, UGA; polypeptide released |
Several ribosomes commonly translate the same mRNA at once, following one another along the message like beads on a string. The assembly is called a polysome, and it is why a cell can produce a great deal of one protein from a single transcript.
What it costs
Protein synthesis is one of the most expensive things a cell does. Loading each amino acid onto its tRNA uses ATP, and each cycle of the ribosome requires further hydrolysis of nucleoside triphosphates to bring the tRNA in and to move the ribosome along. Roughly four high-energy bonds are spent per amino acid added, before any proofreading.
Put a number on it. A modest polypeptide of 300 amino acids therefore costs somewhere near 1200 high-energy bonds, which is why cells that secrete protein heavily — the plasma cells producing antibodies, the acinar cells of the pancreas — are packed with mitochondria as well as with rough endoplasmic reticulum. A question asking why a cell has many mitochondria is often really asking about this.
It also explains why transcription is controlled rather than continuous. There is no sense in producing an mRNA that will be translated thousands of times if the protein is not needed, and the short lifetime of mRNA means that switching a gene off actually stops production within hours.
After the ribosome: folding and finishing
What comes off a ribosome is a polypeptide, which is not yet a protein in the functional sense. It coils and folds into its secondary and tertiary structure, driven by interactions between the side chains — and it starts doing so while it is still being made, as the chain emerges, with the endoplasmic reticulum finishing the job for anything routed into it. Where the protein has quaternary structure the separate chains must then find each other and assemble.
For proteins that will be secreted or inserted into a membrane, the ribosome is attached to the rough endoplasmic reticulum, and the polypeptide is fed into it as it is made. Vesicles then carry it to the Golgi apparatus, where post-translational modification takes place: sugars are added to make glycoproteins, lipids to make lipoproteins, sections may be cut out, and the finished molecule is packaged into a vesicle for export.
Insulin is the standard example, and the numbers are worth following all the way down rather than quoting loosely. It is made as preproinsulin, a single chain of 110 amino acids. The signal sequence of 24 comes off as the chain enters the endoplasmic reticulum, leaving proinsulin at 86. A central connecting section is then cut out: 31 amino acids of connecting peptide plus the four basic residues that flank it and are removed with it, 35 in all. That leaves 86 − 35 = 51, which is exactly the two chains of 21 and 30 amino acids held together by disulfide bridges. The active hormone is under half of what the ribosome originally built.
- Post-translational modification
- Changes made to a polypeptide after translation, such as cutting out sections or adding carbohydrate or lipid groups, largely carried out in the Golgi apparatus. Folding is not one of them: it begins on the ribosome and is finished in the endoplasmic reticulum.
One sequence, all the way through
The examination version of all this is a sequence you are asked to carry from DNA to protein. It is worth doing slowly once so that it can be done quickly under pressure.
From a template strand to an amino acid sequence
The template strand of a short gene reads TAC GGA CTT AAG ATC. Use the codons AUG = methionine, CCU = proline, GAA = glutamic acid, UUC = phenylalanine, UAG = stop. Give the mRNA sequence, the anticodon of the tRNA that delivers proline, and the number of amino acids in the finished polypeptide.
Transcribe first. The mRNA is complementary to the template, with U wherever the template has A: TAC gives AUG, GGA gives CCU, CTT gives GAA, AAG gives UUC and ATC gives UAG. The mRNA is AUG CCU GAA UUC UAG.
The tRNA anticodon is complementary to the codon, not the same as it. Proline's codon here is CCU, so the anticodon is GGA — which happens to match the template triplet, since both are complementary to the same codon.
Now count. There are five codons, but UAG is a stop codon and specifies no amino acid. The polypeptide is therefore four amino acids long: methionine, proline, glutamic acid, phenylalanine. Four amino acids means three peptide bonds and three molecules of water released.
TRY IT — Following a substitution through the whole process
In the gene above, the seventh base of the template strand changes from C to A, so the third triplet reads ATT instead of CTT. The codon GAA codes for glutamic acid and GUA codes for valine. Explain what happens to the mRNA, to the polypeptide, and to the protein's likely function.
Check your answer
Take the transcription step first. The template triplet CTT gave the codon GAA; the altered triplet ATT gives the codon UAA. Working from the template, A pairs with U, T pairs with A and T pairs with A, so the third codon of the mRNA becomes UAA.
UAA is one of the three stop codons. Translation therefore terminates at the third codon rather than the fifth, and the polypeptide released is only two amino acids long — methionine and proline — instead of four.
A polypeptide truncated this early cannot fold into anything useful, so the protein would have no function at all. This is a nonsense mutation, and it is a reminder that a single base substitution is not automatically harmless. Degeneracy protects many third-base changes; it offers no protection when the new codon happens to be a stop signal.
In the exam
- Codons are on mRNA, anticodons on tRNA. If your answer has a tRNA carrying a codon, the mechanism you have described cannot work.
- Name the bond and the reaction: a peptide bond, formed by condensation, releasing water. 'The amino acids join' is not worth a mark.
- Stop codons code for no amino acid, so n codons in a coding sequence give n − 1 amino acids once the stop is counted, and one fewer peptide bond than that.
- Say where each stage happens. Transcription in the nucleus, translation at a ribosome in the cytoplasm or on the rough endoplasmic reticulum, modification in the Golgi apparatus.
- The ribosome moves one codon at a time in the 5′ to 3′ direction along the mRNA. Questions about direction are asking about the mRNA, not the tRNA.
- For energy questions, ATP is used to attach each amino acid to its tRNA and further nucleoside triphosphates are used as the ribosome moves. Both halves of that answer are creditable.
Check yourself
A cell is treated with a drug that prevents tRNA molecules from being loaded with their amino acids, while leaving transcription and the ribosome itself untouched. Predict what happens inside the cell over the next few hours, and explain each part of your prediction.
Answer
Transcription carries on, so mRNA continues to be produced and to leave the nucleus. The ribosomes are intact, so they still bind mRNA and still find start codons. What stops is the supply of loaded tRNA.
An unloaded tRNA can still pair its anticodon with a codon, but it brings no amino acid, so no peptide bond can form and the ribosome cannot move on. Elongation stalls almost immediately, and the polypeptides being made at the moment the drug arrives are left incomplete.
Existing proteins are not destroyed by the drug, so the cell keeps functioning at first. It degrades over hours rather than seconds, as enzymes and other proteins reach the end of their working lives and are broken down without replacement. Proteins with short lifetimes disappear first.
The mRNA also degrades on its own timescale, since it is unstable and nothing about the drug protects it. So the cell ends up with intact DNA, intact ribosomes, a falling stock of mRNA and a falling stock of protein — which is a good illustration of why the loading step, and not the ribosome, is the point at which the genetic code is physically enforced.
Questions
Question 15 marks
Describe what happens at a ribosome from the moment a second tRNA arrives until the ribosome has moved along the mRNA by one codon.
Mark scheme
- B1 a tRNA whose anticodon is complementary to the codon in the A site binds there, held by hydrogen bonds between the paired bases
- B1 that tRNA carries the one specific amino acid corresponding to its anticodon
- B1 the two amino acids are now adjacent, and the ribosome catalyses the formation of a peptide bond between them by condensation, releasing water
- B1 the first tRNA, now without its amino acid, leaves the ribosome
- B1 the ribosome moves along the mRNA by exactly one codon, so the remaining tRNA shifts into the P site and the A site is left free
Question 24 marks
Explain why a plasma cell, which secretes large quantities of antibody, contains a great many mitochondria.
Mark scheme
- B1 the cell synthesises protein continuously and in very large quantities, so translation runs constantly
- B1 ATP is used to attach each amino acid to its tRNA, by the specific enzyme for that amino acid
- B1 further nucleoside triphosphates are hydrolysed to bring each tRNA into the ribosome and to move the ribosome along, roughly four high-energy bonds per amino acid added
- B1 mitochondria supply that ATP by aerobic respiration, so many are needed to keep pace with the demand
Question 34 marks
Some antibiotics stop bacterial protein synthesis by binding to 70S ribosomes, and have no effect on the 80S ribosomes in human cytoplasm. Suggest why such drugs can nevertheless produce side effects in the patient.
Mark scheme
- B1 mitochondria contain their own ribosomes, and these are 70S rather than 80S
- B1 the antibiotic can therefore bind to the ribosomes inside the patient's own mitochondria
- B1 protein synthesis within the mitochondria is inhibited, so the proteins they need cannot be made
- B1 aerobic respiration and the supply of ATP fall, which affects the tissues that demand most ATP
Question 43 marks
The coding sequence of a mature mRNA molecule contains 927 bases and ends with a stop codon. Calculate the number of amino acids in the polypeptide produced and the number of peptide bonds within it.
Mark scheme
- M1 divides the number of bases by three to give the number of codons: 927 ÷ 3 = 309
- M1 subtracts the stop codon, which specifies no amino acid
- A1 308 amino acids, joined by 307 peptide bonds
Question 53 marks
Describe what happens to a polypeptide destined for secretion after it has been released from the ribosome.
Mark scheme
- B1 it folds into its secondary and tertiary structure, a process that begins as the chain emerges and is completed in the endoplasmic reticulum
- B1 vesicles carry it to the Golgi apparatus, where post-translational modification takes place
- B1 carbohydrate or lipid groups may be added and sections may be cut out, and the finished protein is packaged into a vesicle for export
Question 62 marks
Name the site on a ribosome that holds the tRNA carrying the growing polypeptide chain, and name the bond formed between two adjacent amino acids.
Mark scheme
- B1 the P site holds the tRNA carrying the growing polypeptide chain
- B1 a peptide bond is formed between the two amino acids
Worth remembering
- Codons are on mRNA; anticodons are on tRNA; the two pair by hydrogen bonding.
- AUG starts translation and codes for methionine; UAA, UAG and UGA stop it and code for nothing.
- The P site holds the growing chain, the A site accepts the next tRNA, and a peptide bond forms between them by condensation.
- ATP loads each tRNA with its amino acid, and more is spent moving the ribosome along.
- Folding, cutting and the addition of sugars happen after translation, largely in the Golgi apparatus.