D1.2 Protein synthesis
SL & HL+ AHL sectionsSL and HL, with AHL content · Molecules · Theme D
Big questions
- How is a message written in DNA bases turned into a working chain of amino acids?
- What keeps protein production accurate, and what happens when it goes wrong?
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Transcription: making RNA from a DNA template
- RNA polymerase: unwinds the DNA and links RNA nucleotides
- Coding (sense) strand: same base sequence as the RNA, with T in place of U
- Template (antisense) strand: the strand that is copied, read 3′ → 5′
- RNA (here mRNA) leaving the polymerase, 5′ end first
- Free RNA nucleotides (ribonucleoside triphosphates)
- Hydrogen bonds between complementary bases: A–U, T–A, G–C, C–G
- DNA unwinding ahead of the polymerase
- DNA rewinding into a double helix behind it
- Promoter: where RNA polymerase binds to start transcription
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Text description of this visual
Two step-by-step models. In the first, a short gene in double-stranded DNA is transcribed. RNA polymerase binds at the promoter and separates the two strands over a short stretch, forming a bubble. Free RNA nucleotides pair with the bases of the template (antisense) strand by complementary base pairing, held by hydrogen bonds: adenine on the template with uracil, thymine with adenine, guanine with cytosine and cytosine with guanine. The polymerase links them into an RNA strand that grows from its 5′ end to its 3′ end; the 5′ end peels away from the template first and the DNA rewinds behind the polymerase. At the end of the gene the RNA is released. The upper, coding (sense) strand has the same base sequence as the RNA, with thymine in place of uracil. In the second model the same mRNA is translated. It binds to the small subunit of a ribosome, which moves to the start codon AUG. Transfer RNA molecules bring amino acids; each tRNA's anticodon pairs with a complementary codon on the mRNA. The large subunit holds two tRNAs at a time, a peptide bond joins their amino acids, and the ribosome moves along the mRNA one codon at a time towards the 3′ end. A stop codon ends translation and the polypeptide Met–Ala–Phe–Lys–Val is released.
Transcription
- RNA polymerase
- The enzyme that transcribes the gene. It binds to the promoter, separates the DNA strands by breaking the hydrogen bonds between bases, and links RNA nucleotides together into a strand that is complementary to the template.
- Promoter
- A base sequence at the start of the gene where RNA polymerase binds to begin transcription. The promoter itself is not transcribed.
- Unwound DNA (bubble)
- RNA polymerase separates the two DNA strands over a short stretch, so the bases of the template strand are exposed and can pair with RNA nucleotides.
- Coding (sense) strand
- The DNA strand that is not copied. Its base sequence is the same as the RNA's, except that it has thymine (T) where the RNA has uracil (U). It runs 5′→3′ from left to right.
- Template (antisense) strand
- The DNA strand that is copied. It is read 3′→5′ (left to right here), and the RNA made is complementary and antiparallel to it. Its base sequence is not changed by being transcribed.
- Free RNA nucleotides
- RNA nucleotides with ribose and the bases A, U, G or C, carrying two extra phosphates (ribonucleoside triphosphates). Losing those two phosphates provides the energy to link each nucleotide to the chain.
- Complementary base pairing
- Each incoming RNA nucleotide pairs with the next template base. Adenine on the template pairs with uracil in the RNA, thymine with adenine, guanine with cytosine and cytosine with guanine.
- Hydrogen bonds
- Weak bonds between complementary bases (two between A and U or T, three between G and C). They hold each RNA nucleotide in place opposite the right template base while RNA polymerase joins it to the chain.
- Growing 3′ end of the RNA
- Each new nucleotide is joined by its 5′ phosphate to the 3′ end of the growing RNA, so the RNA is made in the 5′→3′ direction.
- mRNA
- The RNA being made. Only about eight of its nucleotides stay paired with the template inside the polymerase; the rest has separated from the DNA. Here the transcript is messenger RNA, which will be translated.
- 5′ end of the mRNA
- The first nucleotide to be added. It is the first part of the RNA to leave the polymerase.
- DNA rewinds
- Behind the polymerase the two DNA strands pair up again and re-form the double helix. The DNA is unchanged, so the gene can be transcribed again and again.
- End of the gene
- When RNA polymerase reaches the end of the gene, it releases the RNA and detaches from the DNA.
- RNA polymerase binds at the promoter. RNA polymerase binds to the DNA at the promoter, a sequence at the start of the gene. Only genes whose promoter the polymerase binds to are transcribed, so this is a key point at which a gene can be switched on or off.
- The DNA strands separate. RNA polymerase breaks the hydrogen bonds between base pairs and separates the two strands over a short stretch (a transcription bubble). The bases of the template strand are now exposed. The other strand, the coding strand, is not copied.
- Complementary base pairing. Free RNA nucleotides diffuse to the active site and pair with the exposed template bases by hydrogen bonding, A with U, T with A, G with C and C with G. RNA polymerase links each one to the chain with a covalent bond (between sugar and phosphate). Uracil is used in RNA in place of thymine.
- The RNA grows 5′ → 3′. RNA polymerase moves along the template strand, adding nucleotides one at a time to the 3′ end of the RNA. The RNA separates from the template as it leaves the polymerase, 5′ end first, and the DNA rewinds behind it. The RNA has the same sequence as the coding strand, with U instead of T.
- The RNA is released. At the end of the gene, RNA polymerase releases the completed RNA and detaches. The DNA closes into a double helix again with its base sequence unchanged, so it can be used as a template many more times. In eukaryotes this pre-mRNA is then modified before it leaves the nucleus.
Translation
- mRNA
- Messenger RNA carrying the base sequence copied from the gene. It is read in codons (triplets of bases) from the 5′ end towards the 3′ end. This is the mRNA made in the transcription model.
- 5′ end of the mRNA
- The ribosome binds near the 5′ end of the mRNA and moves towards the 3′ end, so translation proceeds 5′→3′.
- Small ribosomal subunit
- The mRNA binds to the small subunit. Ribosomes are made of ribosomal RNA (rRNA) and protein, in two subunits that come together on an mRNA.
- Large ribosomal subunit
- Joins once the start codon is found. It has binding sites for tRNA, so two tRNAs can be bound at the same time, and its rRNA catalyses peptide bond formation.
- Start codon (AUG)
- Translation starts at the first AUG codon. AUG codes for methionine, so polypeptides start with methionine (which is sometimes removed later).
- Codon
- A sequence of three bases on the mRNA that codes for one amino acid. This codon, GCA, codes for alanine.
- Anticodon
- Three bases on a tRNA that are complementary to a codon on the mRNA. They pair antiparallel by hydrogen bonds; for example the anticodon 3′-UAC-5′ pairs with the codon 5′-AUG-3′.
- Transfer RNA (tRNA)
- A folded RNA molecule with an anticodon at one end and a specific amino acid attached at the other. Each tRNA brings the amino acid that matches the codon its anticodon pairs with.
- A site
- Where each tRNA carrying a new amino acid binds, if its anticodon matches the codon there.
- P site
- Holds the tRNA carrying the growing polypeptide. The initiator tRNA starts here. The chain is transferred from this tRNA to the amino acid on the A-site tRNA.
- E site
- The exit site. After translocation the tRNA that has given up its amino acid moves here, then leaves the ribosome to be reloaded with another amino acid of its kind.
- Peptide bond
- A covalent bond between the carboxyl group of one amino acid and the amine group of the next, made by a condensation reaction. It is catalysed by rRNA in the large subunit.
- Growing polypeptide
- The chain of amino acids, in the order set by the codons. It grows by one amino acid per codon and leaves the ribosome through a tunnel in the large subunit.
- Stop codon (UAA)
- UAA, UAG and UGA are stop codons. No tRNA has an anticodon for them, so when one reaches the A site, translation stops and the polypeptide is released.
- Release factor
- A protein (not a tRNA) that binds in the A site when a stop codon arrives there. It causes the bond between the polypeptide and the last tRNA to be broken, so the chain is released.
- mRNA binds to the small subunit. The small subunit of a ribosome, already carrying an initiator tRNA, binds to the mRNA near its 5′ end. The ribosome will read the mRNA from 5′ to 3′.
- Start codon and large subunit. The small subunit moves along the mRNA to the first AUG, the start codon. The anticodon UAC of the initiator tRNA pairs with it, bringing methionine. The large subunit then joins, completing the ribosome.
- A tRNA brings the next amino acid. A tRNA whose anticodon (CGU) is complementary to the next codon (GCA) binds to the ribosome, carrying alanine. Complementary base pairing between codon and anticodon makes sure the right amino acid is brought. Two tRNAs are now bound at once.
- A peptide bond forms. The ribosome joins methionine to alanine by a peptide bond. The growing chain is now attached to the newest tRNA, and the first tRNA no longer carries an amino acid. The reaction is catalysed by rRNA in the large subunit.
- The ribosome moves one codon. The ribosome moves three bases (one codon) along the mRNA towards the 3′ end. The tRNA carrying the chain moves to the middle site, the empty tRNA moves to the exit site, and the next codon (UUC) is exposed.
- Elongation repeats. The cycle repeats for each codon — a tRNA with the matching anticodon binds, a peptide bond joins its amino acid to the chain, and the ribosome moves one codon on. Each empty tRNA leaves and can pick up another amino acid. Phenylalanine, lysine and valine are added in turn.
- Stop codon — the polypeptide is released. The stop codon UAA reaches the ribosome. No tRNA pairs with it; a release factor protein binds instead, the bond holding the chain to the last tRNA is broken, and the polypeptide Met–Ala–Phe–Lys–Val is released. The subunits separate and can translate another mRNA.
The information in a gene is stored as a sequence of bases in DNA. To use it, the cell first copies the sequence into RNA. Transcription is the synthesis of RNA using DNA as a template. It happens in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotes.
The roles of RNA polymerase
The enzyme RNA polymerase carries out every stage:
- It binds to the DNA at the start of a gene, at a sequence called the promoter.
- It separates the two DNA strands over a short stretch by breaking the hydrogen bonds between the bases, exposing the bases of one strand.
- It moves along that strand, the template strand, and links RNA nucleotides that have paired with the template bases, forming covalent bonds between the ribose of one nucleotide and the phosphate of the next.
- At the end of the gene it releases the RNA and detaches.
Only one strand is copied. The RNA is complementary to the template (antisense) strand, so it has the same base sequence as the other strand, the coding (sense) strand, except that RNA has uracil (U) where DNA has thymine (T). No primer is needed.
Hydrogen bonds and complementary base pairing
The free nucleotides in the nucleoplasm carry the bases A, U, G and C. Each one can only form stable hydrogen bonds with one kind of exposed template base, so the RNA sequence is set by the template:
| Base on the DNA template | Base in the RNA |
|---|---|
| A (adenine) | U (uracil) |
| T (thymine) | A (adenine) |
| G (guanine) | C (cytosine) |
| C (cytosine) | G (guanine) |
The hydrogen bonds hold each nucleotide in position while RNA polymerase joins it to the chain. Behind the polymerase they break again: the RNA peels away from the template and the two DNA strands re-form the double helix.
Stable templates and switching genes on
DNA templates are not changed
A single DNA strand can be used as a template again and again. During transcription the two strands are separated only by breaking hydrogen bonds, which re-form behind the polymerase. The covalent sugar–phosphate backbone and the base sequence of the template are left exactly as they were. A gene that is needed in large amounts can therefore be transcribed many times, often by several RNA polymerases one behind another.
This stability matters most in somatic cells that do not divide, such as most neurons and heart muscle cells. They cannot replace damaged DNA by making a new copy from a dividing parent cell, so the base sequences of their genes must be conserved for the whole life of the cell, which may be many decades. DNA repair enzymes help by correcting damage to the template.
Transcription is the first stage of gene expression
Gene expression means using the information in a gene to make its product, usually a protein. Its first stage is transcription, and the second is translation.
- Not all genes are expressed at any one time. A liver cell and a skin cell contain the same genes, but they transcribe different sets of them, and a cell changes which genes it transcribes as conditions change.
- A gene that is not transcribed makes no mRNA, so its protein cannot be made. Because transcription comes first, it is a key point at which genes are switched on or off, for example by controlling whether RNA polymerase can bind to the promoter.
Regulation can also happen later, but controlling transcription avoids wasting energy and nucleotides on RNA that is not needed. How cells control gene expression is covered in D2.2 Gene expression.
Translation: mRNA, ribosomes and tRNA
Translation is the synthesis of a polypeptide using the base sequence of mRNA. The sequence of bases is “translated” into a sequence of amino acids, in the same order. It happens on ribosomes in the cytoplasm (free, or bound to the rough endoplasmic reticulum).
Three kinds of RNA, three roles
| Component | Role in translation |
|---|---|
| mRNA (messenger RNA) | Carries the base sequence of a gene from the DNA to the ribosome; read in codons of three bases |
| Ribosome (rRNA + protein) | Brings mRNA and tRNAs together and catalyses peptide bonds between amino acids |
| tRNA (transfer RNA) | Carries a specific amino acid and has an anticodon that pairs with the matching codon |
A ribosome has two subunits. The mRNA binds to the small subunit. The large subunit has binding sites for tRNA, so two tRNAs can be bound at the same time, each paired with an adjacent codon. This lets the ribosome hold the growing chain on one tRNA while the next amino acid arrives on another. The large subunit’s ribosomal RNA also catalyses the peptide bond.
Codon–anticodon pairing
Each tRNA molecule is a single RNA strand folded into a precise shape. At one end it has an anticodon, three bases complementary to one codon; at the other end it carries the amino acid that codon specifies. Enzymes load each tRNA with the right amino acid.
On the ribosome, a tRNA stays bound only if its anticodon forms complementary base pairs with the codon, using hydrogen bonds (A–U and G–C). Codon and anticodon are antiparallel: the codon 5′-GCA-3′ pairs with the anticodon 3′-CGU-5′. This pairing is what makes sure the right amino acid is added each time.
The genetic code and codon tables
The genetic code is the set of rules by which the base sequence of mRNA is translated into an amino acid sequence.
Why a triplet code?
There are four different bases and twenty amino acids. A code using one base per amino acid gives only 4 combinations, and two bases only 4 × 4 = 16, which is still too few. Three bases give 4 × 4 × 4 = 64 codons, enough for all twenty amino acids. So each codon is a triplet of bases, read one after another without gaps or overlaps.
Features of the code
- Degenerate: there are 64 codons for 20 amino acids, so most amino acids have more than one codon. Leucine, serine and arginine have six each; only methionine and tryptophan have one. Codons for the same amino acid often differ only in the third base, so some base substitutions do not change the protein.
- Start and stop: AUG codes for methionine and is also the start codon. UAA, UAG and UGA are stop codons, which code for no amino acid. That leaves 61 codons for amino acids.
- Universal: almost every organism, from bacteria to humans, uses the same codons for the same amino acids. There are a few minor variations, for example in mitochondria. This is why bacteria given a copy of the human insulin gene (without its introns) can make human insulin, and it is strong evidence that all life shares a common origin.
Using a codon table
To deduce an amino acid sequence:
- If you are given DNA, convert it to mRNA first (pair the template, or copy the coding strand with U for T).
- Find the start codon (AUG) if the question involves one, then split the mRNA into triplets from there.
- Look up each codon: first base in the row, second base in the column, third base within the box.
- Stop at the first stop codon; it adds nothing to the chain.
DNA coding strand5′ ATGGCATTCAAAGTGTAA 3′
DNA template strand3′ TACCGTAAGTTTCACATT 5′
mRNA5′ AUGGCAUUCAAAGUGUAA 3′
- 1AUGUACMet
- 2GCACGUAla
- 3UUCAAGPhe
- 4AAAUUULys
- 5GUGCACVal
- 6UAAAUUSTOP
codon (mRNA, 5′→3′) anticodon (tRNA, 3′→5′) amino acid
Reading from the start codon at base 1: 6 codons → Met–Ala–Phe–Lys–Val (5 amino acids; the stop codon UAA ends translation).
| 1st base ↓ 2nd base → | U | C | A | G |
|---|---|---|---|---|
| U | ||||
| C | ||||
| A | ||||
| G |
Standard genetic code. Each codon is read 5′→3′ on the mRNA; within each cell the third base runs U, C, A, G from top to bottom. AUG codes for methionine and is also the start codon; UAA, UAG and UGA are stop codons and code for no amino acid.
Text description of this simulation
A decoder for the standard genetic code. You type a sequence as mRNA (5′→3′), as the DNA coding strand (5′→3′) or as the DNA template strand written 3′→5′, or add codons by picking them from a 64-codon table arranged by first, second and third base. The tool shows the coding strand, template strand and mRNA lined up, splits the mRNA into codons, and lists the tRNA anticodon and amino acid for each. Reading starts at the first AUG start codon, or at base 1, 2 or 3 to compare reading frames, and stops at the first stop codon (UAA, UAG or UGA). An example from the human β-globin gene can be switched to the sickle-cell allele, where one base substitution (GAG to GUG in the mRNA) replaces glutamic acid with valine at position 6.
Ribosome movement and peptide bonds
Once a ribosome has assembled at the start codon, the polypeptide grows by repeating the same cycle for every codon. This stage is elongation.
- A tRNA with an anticodon complementary to the next codon binds to the ribosome, carrying its amino acid. Two tRNAs are now bound side by side, each paired with a codon.
- The amino acid (or growing chain) on the first tRNA is transferred to the amino acid on the new tRNA, and they are joined by a peptide bond. This is a condensation reaction between the carboxyl group of one amino acid and the amine group of the next. It is catalysed by the ribosome, in fact by its rRNA.
- The ribosome moves along the mRNA by one codon (three bases). The tRNA carrying the chain shifts along with its codon, and the empty tRNA is released to collect another amino acid.
- The next codon is now in position and the cycle repeats.
The ribosome therefore moves along the mRNA stepwise, one codon at a time, and the chain grows by one amino acid per step. The order of codons on the mRNA determines the order of amino acids in the polypeptide, its primary structure (see B1.2 Proteins).
When a stop codon reaches the ribosome, no tRNA can pair with it. The finished polypeptide is released, and the ribosome subunits separate.
Mutations that change protein structure
A mutation is a change in the base sequence of DNA. Because the sequence of codons determines the amino acid sequence, a mutation in a gene can change the protein it codes for. A point mutation affects a single base. In a base substitution, one base is replaced by another, which changes one codon. The effect depends on the new codon:
- it may code for the same amino acid (thanks to degeneracy), so the protein is unchanged;
- it may code for a different amino acid, which can change how the polypeptide folds and functions;
- it may become a stop codon, so the polypeptide is shorter and usually does not work.
Sickle cell anaemia
The best-known example is in the gene for the β-globin polypeptide of haemoglobin (HBB). In the sickle-cell allele (HbS) one base is substituted in the codon for the sixth amino acid:
- DNA coding strand: GAG → GTG (template CTC → CAC)
- mRNA codon: GAG → GUG
- amino acid 6: glutamic acid → valine
Glutamic acid has a polar, negatively charged R group; valine’s is non-polar. The non-polar valine on the surface of haemoglobin S makes the molecules stick together into long fibres when the oxygen concentration is low. The fibres distort red blood cells into a rigid sickle shape, and the cells block capillaries and are destroyed early, causing anaemia. Try the sickle-cell switch in the codon-table simulation above.
Directionality of transcription and translation
Every nucleic acid strand has a 5′ end (with a phosphate on carbon 5′ of the sugar) and a 3′ end (with a hydroxyl group on carbon 3′). Both stages of protein synthesis work in one direction only.
5′ → 3′ transcription
RNA polymerase can only add a nucleotide to the 3′ end of the growing RNA. The 5′ phosphate of the incoming nucleotide is linked to the free 3′–OH of the last nucleotide in the chain. The RNA is therefore synthesized in the 5′ → 3′ direction. Because the RNA is antiparallel to the template strand, the polymerase moves along the template from its 3′ end towards its 5′ end.
5′ → 3′ translation
The ribosome binds near the 5′ end of the mRNA and moves towards the 3′ end, translating one codon at a time. So translation also proceeds 5′ → 3′. The first amino acid, methionine, ends up at the free amino end of the polypeptide (the N-terminus), and each new amino acid is added at the carboxyl end (the C-terminus).
Initiation of transcription at the promoter
Each gene has a promoter, a base sequence just upstream of (before) the point where transcription starts. The promoter is where transcription is initiated, but it is not itself transcribed and does not code for any amino acids.
- In prokaryotes, RNA polymerase can bind directly to the promoter.
- In eukaryotes, RNA polymerase cannot bind on its own. Proteins called transcription factors must first bind to the promoter (and to nearby sequences). The polymerase then binds to this complex, separates the DNA strands at the start point and begins transcription, moving away from the promoter along the gene.
Several transcription factors are usually needed, and some only bind after they have been activated, for example by a hormone. Other proteins (repressors) can bind and prevent transcription. Because different genes have different promoter sequences and need different combinations of factors, a cell can switch some genes on while leaving others off. You do not need to name specific transcription factors.
Non-coding sequences in DNA
Only a small fraction of eukaryotic DNA codes for polypeptides; in humans it is roughly 1–2% of the genome. Many other sequences have functions even though they are never translated. Four kinds are required:
- Regulators of gene expression: promoters, and enhancers and silencers further away. Transcription factors and other proteins bind to them to control whether, and how much, a gene is transcribed.
- Introns: sequences within genes that are transcribed into pre-mRNA but removed before translation. Exons are the parts that remain.
- Telomeres: repeated short sequences at the ends of chromosomes (TTAGGG repeated hundreds or thousands of times in humans). They protect the coding DNA near the ends, because a little of the end is lost each time the DNA is replicated.
- Genes for tRNA and rRNA: these genes are transcribed, but the RNA made is used directly, as transfer RNA or as ribosomal RNA, without being translated. Eukaryotes have many copies of the rRNA genes, so ribosomes can be made quickly.
Processing pre-mRNA and alternative splicing
In eukaryotes, transcription produces pre-mRNA (the primary transcript), which is modified in the nucleus before it is exported to the cytoplasm. Prokaryotes do not do this: their mRNA can be translated while it is still being made, and most of their genes have no introns.
Post-transcriptional modification
- 5′ cap: a modified guanine nucleotide is added to the 5′ end.
- Poly-A tail: a long chain of adenine nucleotides (often 100–250) is added to the 3′ end.
- Splicing: the introns are cut out and the exons are joined together, giving a continuous coding sequence. The removed introns are broken down in the nucleus.
The cap and tail stabilize the mRNA, protecting it from enzymes that break RNA down from its ends, so it lasts longer and can be translated more times. The cap also helps the ribosome bind. The finished molecule is mature mRNA.
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Text description of this animation
A pre-mRNA in the nucleus of a eukaryotic cell, with four exons separated by three introns, shown in five steps. A 5′ cap is added to the 5′ end and a poly-A tail to the 3′ end. The introns are cut out and the exons are spliced together to form mature mRNA. Finally, alternative splicing joins a different combination of exons from the same pre-mRNA, leaving out exon 3, so the same gene gives two different polypeptides.
- Pre-mRNA. Transcription in the nucleus of a eukaryotic cell produces pre-mRNA (the primary transcript). It contains exons, the sequences that will be translated, separated by introns, which will not.
- 5′ cap added. A modified guanine nucleotide, the 5′ cap, is added to the 5′ end. It protects the mRNA from being broken down and helps the ribosome bind to it.
- Poly-A tail added. A long chain of adenine nucleotides, the poly-A tail, is added to the 3′ end. It also protects the mRNA from breakdown, so the mRNA lasts longer in the cytoplasm and can be translated more times.
- Introns removed, exons joined. The introns are cut out and the exons are joined together (splicing). The result is mature mRNA with a continuous coding sequence, which leaves the nucleus through a nuclear pore.
- Alternative splicing. Splicing can join different combinations of exons from the same pre-mRNA. Here exon 3 is left out of the second mature mRNA. Each version is translated into a different polypeptide, so one gene can code for several protein variants.
Alternative splicing
The exons of one pre-mRNA do not always have to be joined in the same way. By including some exons and leaving out others, a cell can make different mature mRNAs from a single gene, which are translated into different polypeptides: variants of a protein. This is called alternative splicing. It is one reason why humans make many more different proteins than they have genes (about 20 000 protein-coding genes). Which variant is made can differ between tissues or with conditions. You do not need specific examples.
Initiation of translation and the A, P and E sites
Translation begins with the assembly of a ribosome at the start codon:
- The small ribosomal subunit attaches to the 5′ end of the mRNA (in eukaryotes it recognizes the 5′ cap).
- It moves along the mRNA towards the 3′ end until it reaches the start codon, AUG.
- The initiator tRNA, with anticodon UAC and carrying methionine, pairs with the start codon.
- The large subunit attaches, completing the ribosome, with the initiator tRNA in the P site.
- Another tRNA with the anticodon for the second codon binds in the A site, and elongation begins.
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Text description of this animation
An mRNA with a 5′ cap, read 5′→3′ from left to right, shown in seven steps. The small ribosomal subunit, carrying the initiator tRNA with methionine, binds near the 5′ end and moves along to the start codon AUG, where the anticodon UAC pairs with it. The large subunit joins, so the initiator tRNA is in the P site. A second tRNA carrying alanine enters the A site; a peptide bond joins methionine to alanine; the ribosome moves one codon towards the 3′ end, so the empty tRNA is in the E site and the dipeptide-carrying tRNA in the P site. The empty tRNA then leaves and the next tRNA enters the A site.
- Small subunit binds the 5′ end. The small subunit of the ribosome, carrying the initiator tRNA (anticodon UAC, with methionine attached), attaches to the 5′ end of the mRNA. In eukaryotes it recognizes the 5′ cap.
- Moves to the start codon. The small subunit moves along the mRNA towards the 3′ end until it reaches the first AUG, the start codon. The anticodon of the initiator tRNA pairs with AUG by complementary base pairing.
- Large subunit attaches. The large subunit attaches, completing the ribosome. It has three tRNA binding sites, E, P and A. The initiator tRNA is in the P site and the A site is over the next codon, GCA.
- A second tRNA enters the A site. A tRNA with the anticodon CGU, complementary to GCA, binds in the A site, carrying alanine. A tRNA with any other anticodon would not pair with the codon and would leave again.
- Peptide bond in the large subunit. Methionine is detached from the tRNA in the P site and joined to alanine by a peptide bond, catalysed by rRNA in the large subunit. The chain is now held by the tRNA in the A site.
- Translocation. The ribosome moves one codon along the mRNA towards the 3′ end. The empty initiator tRNA is now in the E site, the tRNA carrying the dipeptide is in the P site, and the A site is empty, over the next codon UUC.
- E-site tRNA leaves; the cycle repeats. The empty tRNA leaves from the E site and can be loaded with another methionine. A tRNA with the anticodon AAG enters the A site with phenylalanine, and the cycle of peptide bond formation and translocation repeats.
The three tRNA binding sites
| Site | Role during elongation |
|---|---|
| A (aminoacyl) | A tRNA carrying a new amino acid binds here, if its anticodon matches the codon |
| P (peptidyl) | Holds the tRNA attached to the growing polypeptide; the chain is transferred from here to the amino acid on the A-site tRNA |
| E (exit) | The empty tRNA moves here after translocation, then leaves the ribosome |
After each peptide bond forms, the ribosome moves one codon towards the 3′ end (translocation): the tRNA in the A site moves to the P site, the empty tRNA in the P site moves to the E site and leaves, and the A site is free for the next tRNA.
Modifying polypeptides into their functional state
The polypeptide released from a ribosome is often not yet a working protein. It may need to be folded, cut, joined to other polypeptides, or have non-amino-acid groups attached, such as carbohydrate chains in the Golgi apparatus. Many proteins are made as longer, inactive precursors that are activated by removing part of the chain.
Pre-proinsulin to insulin
Insulin, the hormone secreted by β cells of the pancreas, is the required example. It is modified in two stages:
- Pre-proinsulin (110 amino acids) is made by ribosomes on the rough ER. Its N-terminal signal peptide (24 amino acids) directs it into the rough ER, where the signal peptide is cut off. The result is proinsulin (86 amino acids). Proinsulin folds and forms three disulfide bonds.
- Proinsulin is carried to the Golgi apparatus and packaged into secretory vesicles, where protease enzymes remove the C peptide (31 amino acids, plus two amino acids at each junction). What remains is insulin: an A chain of 21 amino acids and a B chain of 30, held together by two disulfide bonds, with a third bond within the A chain.
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Text description of this animation
The modification of the polypeptide made by translating the insulin gene, in four steps. Pre-proinsulin, 110 amino acids long, consists of a signal peptide, the B chain, the C peptide and the A chain. In the rough endoplasmic reticulum the signal peptide is cut off, leaving proinsulin with 86 amino acids. Proinsulin folds and three disulfide bonds form: two between the future A and B chains and one within the A chain. In the Golgi apparatus and secretory vesicles, proteases remove the C peptide, leaving insulin: an A chain of 21 amino acids and a B chain of 30, held together by the two disulfide bonds.
- Pre-proinsulin is made. Translation of the insulin mRNA by a ribosome on the rough ER produces pre-proinsulin, 110 amino acids long. At its N-terminal end is a signal peptide of 24 amino acids that directs the chain into the rough ER.
- Stage 1 — signal peptide removed. Inside the rough ER, an enzyme cuts off the signal peptide. The remaining polypeptide, 86 amino acids long, is proinsulin.
- Proinsulin folds. Proinsulin folds into its three-dimensional shape, and three disulfide bonds form between cysteine R groups — two linking the future A and B chains and one within the A chain. The C peptide connects the B chain to the A chain.
- Stage 2 — C peptide removed. In the Golgi apparatus and the secretory vesicles that bud from it, proteases cut out the C peptide. What remains is insulin, an A chain (21 amino acids) and a B chain (30) held together by two disulfide bonds. Insulin is stored in vesicles until it is secreted by exocytosis.
Insulin is then stored in the vesicles until it is released by exocytosis. Making it as an inactive precursor allows it to fold correctly before it is cut, which is how the A and B chains end up correctly linked.
Recycling amino acids by proteasomes
The proteome is the full set of proteins a cell makes at a given time. Keeping it working needs not only constant protein synthesis but also constant protein breakdown:
- proteins that are damaged or misfolded would not work and might stick together, so they must be removed;
- proteins that are no longer needed, such as enzymes or regulatory proteins whose job is done, must be removed so the cell can change what it is doing.
Proteins for breakdown are tagged with a chain of small proteins called ubiquitin. Proteasomes, large barrel-shaped complexes of protease enzymes, recognize the tag, unfold the protein using energy from ATP, and cut it into short peptides inside the barrel. The ubiquitin is released and reused. The peptides are broken down further into amino acids, which are returned to the pool used for translation.
Recycling amino acids in this way saves the energy and resources that would be needed to obtain new ones. The balance between synthesis and breakdown sets the amount of each protein in the cell.
Must-know definitions
- Transcription
- The synthesis of RNA using one strand of DNA, the template strand, as a guide. RNA polymerase separates the DNA strands and links RNA nucleotides that pair with the template bases, making an RNA with the same sequence as the coding strand (U in place of T).
- Complementary base pairing in transcription
- Free RNA nucleotides are held opposite template bases by hydrogen bonds: A on the template pairs with U, T with A, G with C and C with G. This makes the RNA base sequence an exact complement of the template.
- Translation
- The synthesis of a polypeptide on a ribosome, in which the base sequence of mRNA, read as codons, sets the sequence of amino acids. mRNA binds to the small subunit; tRNAs bring the amino acids.
- Codon and anticodon
- A codon is a sequence of three bases on mRNA that codes for one amino acid (or stop). An anticodon is the complementary triplet on a tRNA; codon and anticodon pair by hydrogen bonds.
- Degeneracy of the genetic code
- Most amino acids are coded for by more than one codon (for example leucine by six). There are 64 codons but only 20 amino acids, so the code has redundancy.
- Universality of the genetic code
- Almost all organisms use the same codons for the same amino acids, with only a few minor variations (for example in mitochondria). This is evidence of common ancestry.
- Point mutation (base substitution)
- A change in a single base in DNA. It can change one codon and so one amino acid, as in sickle cell anaemia, where GAG becomes GTG in the coding strand and glutamic acid is replaced by valine.
- Promoter and transcription factorsAHL
- The promoter is a non-transcribed DNA sequence at the start of a gene. In eukaryotes, transcription factors (proteins) bind to it, allowing RNA polymerase to bind and begin transcription.
- Post-transcriptional modificationAHL
- Changes to eukaryotic pre-mRNA in the nucleus: a 5′ cap and a 3′ poly-A tail are added, which stabilize the mRNA, and introns are removed while exons are spliced together.
- Alternative splicingAHL
- Joining different combinations of exons from the same pre-mRNA, so that one gene can code for more than one polypeptide.
- ProteasomeAHL
- A large barrel-shaped complex of protease enzymes that breaks down proteins tagged with ubiquitin into short peptides, so their amino acids can be reused. Constant breakdown and synthesis maintain a working proteome.
Common exam mistakes
Loses marks RNA polymerase copies both strands of the DNA.
Write instead Only one strand, the template (antisense) strand, is transcribed. The RNA has the same base sequence as the other, coding (sense) strand, except that it has U where the coding strand has T.
Loses marks The mRNA has the same sequence as the template strand.
Write instead The mRNA is complementary to the template strand. It matches the coding strand (with U for T).
Loses marks Adenine on the DNA template pairs with thymine in the RNA.
Write instead RNA contains uracil, not thymine, so adenine on the template pairs with uracil. Thymine on the template pairs with adenine.
Loses marks Transcription uses up or damages the DNA, so a gene can only be copied a few times.
Write instead The DNA strands separate only temporarily, and the base sequence of the template is not changed. It rewinds afterwards and can be transcribed many times — which matters in cells such as neurons that never divide.
Loses marks Each amino acid has exactly one codon.
Write instead The code is degenerate: most amino acids have two, three, four or six codons. Only methionine (AUG) and tryptophan (UGG) have just one.
Loses marks tRNA anticodons are found on the mRNA; codons are on the tRNA.
Write instead Codons are on the mRNA and anticodons are on tRNA. When using a codon table, always look up the mRNA codon, not the anticodon or the DNA triplet.
Loses marks Stop codons code for a special amino acid that ends the chain.
Write instead Stop codons (UAA, UAG, UGA) code for no amino acid. No tRNA pairs with them; a release factor binds and the polypeptide is released.
Loses marks In sickle cell anaemia, valine is replaced by glutamic acid.
Write instead The direction is the other way: glutamic acid (polar, charged) at position 6 of β-globin is replaced by valine (non-polar), because GAG becomes GUG in the mRNA.
Loses marks Transcription runs 3′ → 5′ because the template is read 3′ → 5′.
Write instead The RNA is made 5′ → 3′: each nucleotide is added to the 3′ end of the chain. The template is read 3′ → 5′ because the two are antiparallel.
AHLLoses marks Introns are removed from the DNA before transcription.
Write instead Introns stay in the DNA and are transcribed into pre-mRNA. They are removed from the RNA by splicing in the nucleus, after transcription.
AHL
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Multiple-choice questions
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What are the roles of RNA polymerase in transcription?
It binds to the promoter, separates the DNA strands by breaking hydrogen bonds between bases, and links RNA nucleotides that pair with the template strand into an RNA strand (5′→3′). It releases the RNA at the end of the gene.
Template strand vs coding strand
The template (antisense) strand is the one transcribed; the RNA is complementary to it. The coding (sense) strand is not transcribed; it has the same sequence as the RNA, except T instead of U.
Which RNA base pairs with adenine on the DNA template?
Uracil (U). RNA has no thymine. The full set is A→U, T→A, G→C, C→G, held by hydrogen bonds.
Why can a DNA template be transcribed many times?
Its strands separate only by breaking hydrogen bonds, which re-form afterwards. The base sequence is not changed. This matters in non-dividing cells, which must keep the same sequences all their life.
Why is transcription a key control point for gene expression?
It is the first stage of gene expression. Not all genes are expressed at once; a gene that is not transcribed produces no mRNA, so its protein cannot be made.
Roles of mRNA, ribosomes and tRNA in translation
mRNA carries the codons and binds to the small ribosomal subunit. tRNA brings a specific amino acid and pairs its anticodon with a codon. The ribosome holds two tRNAs at once on its large subunit and catalyses peptide bonds.
Codon vs anticodon
A codon is three bases on mRNA coding for one amino acid. An anticodon is the complementary three bases on tRNA. They pair antiparallel, e.g. codon 5′-AUG-3′ with anticodon 3′-UAC-5′.
Why is the genetic code a triplet code?
Four bases taken two at a time give only 16 combinations, fewer than the 20 amino acids. Triplets give 4³ = 64 codons, enough for all amino acids and stop signals.
What do "degenerate" and "universal" mean for the genetic code?
Degenerate — most amino acids have more than one codon (64 codons, 20 amino acids). Universal — almost all organisms use the same codons for the same amino acids, with a few minor exceptions.
Start and stop codons
AUG is the start codon and codes for methionine. UAA, UAG and UGA are stop codons and code for no amino acid.
What happens in each cycle of elongation?
A tRNA with a matching anticodon binds, a peptide bond joins its amino acid to the growing chain (catalysed by rRNA), and the ribosome moves one codon along the mRNA towards the 3′ end. The empty tRNA leaves.
The sickle-cell mutation — bases and amino acids
A base substitution in the β-globin gene. Coding strand GAG → GTG; mRNA GAG → GUG; amino acid 6 glutamic acid → valine. HbS forms fibres at low oxygen and red blood cells become sickle-shaped.
What does 5′ → 3′ mean for transcription and for translation?
Transcription — nucleotides are added to the 3′ end of the RNA, so it is built 5′→3′ (the template is read 3′→5′). Translation — the ribosome moves along the mRNA from its 5′ end to its 3′ end.
How is transcription initiated in eukaryotes?
Transcription factors bind to the promoter, a non-transcribed sequence upstream of the gene. RNA polymerase then binds and begins transcription at the start point.
Name four kinds of non-coding DNA sequence.
Regulators of gene expression (promoters, enhancers), introns, telomeres, and genes for tRNA and rRNA (transcribed but not translated).
Three modifications of eukaryotic pre-mRNA
A 5′ cap and a 3′ poly-A tail are added (they stabilize the mRNA), and introns are removed while exons are spliced together. Alternative splicing joins different exon combinations, giving several polypeptides from one gene.
Roles of the A, P and E sites
A — a tRNA carrying the next amino acid binds. P — holds the tRNA with the growing chain (the initiator tRNA starts here). E — the empty tRNA exits after translocation.
How is pre-proinsulin turned into insulin, and how are amino acids recycled?
The signal peptide is removed in the rough ER (→ proinsulin), then the C peptide in the Golgi/vesicles (→ insulin, A + B chains joined by disulfide bonds). Unneeded proteins are tagged with ubiquitin and digested by proteasomes; their amino acids are reused.
Last reviewed 19 October 2026. Spotted a mistake? Report it.