How Protein Synthesis Works: Transcription, Translation and Mutations

From gene to protein

A gene is a stretch of DNA whose sequence of bases spells out a protein. The cell does not read the DNA directly to make the protein. It first copies the gene into messenger RNA (transcription), then ribosomes read the mRNA to join amino acids in the right order (translation). This flow of information, DNA to RNA to protein, is often called the central dogma of molecular biology.

Transcription

RNA polymerase binds at a promoter in front of the gene, unwinds a short stretch of the double helix (the transcription bubble, about 14 base pairs) and moves along it. It reads one strand, the template strand, and builds an RNA strand that is its complement, joining nucleotides to the RNA's 3′ end so it grows 5′ to 3′. The RNA therefore has the same sequence as the other DNA strand, the coding strand, with uracil (U) in place of thymine (T). Behind the polymerase the DNA zips back up.

In bacteria the mRNA is used at once. In eukaryotes it is processed first: a cap is added to the 5′ end, a poly-A tail to the 3′ end, and the introns are cut out (splicing) before the mRNA leaves the nucleus.

The genetic code

The mRNA is read three bases at a time. Each triplet is a codon. Four bases make 4³ = 64 codons: 61 code for the 20 amino acids and 3 (UAA, UAG, UGA) mean stop. AUG codes for methionine and also marks the start. Most amino acids have several codons, usually differing in the third base, so the code is redundant (degenerate). The same code is used, with minor exceptions, by almost every living thing.

Translation

1. Initiation: the small subunit of the ribosome binds the mRNA and finds the start codon AUG; the first transfer RNA, carrying methionine, pairs with it, and the large subunit joins.
2. Elongation: a tRNA whose anticodon pairs with the next codon brings the next amino acid; the ribosome joins it to the growing chain with a peptide bond, then moves on by one codon. Bacterial ribosomes add about 15 to 20 amino acids a second, ours about 5.
3. Termination: at a stop codon no tRNA fits; a release factor binds instead, the finished chain is let go, and the ribosome comes apart.

The order of codons fixes the order of amino acids, and that sequence decides how the chain folds into a working protein.

A worked example: β-globin

The human β-globin gene (HBB), half of the hemoglobin molecule, begins

ATG GTG CAT CTG ACT CCT GAG GAG AAG …

1. mRNA: AUG GUG CAU CUG ACU CCU GAG GAG AAG …
2. Codons: AUG = Met, GUG = Val, CAU = His, CUG = Leu, ACU = Thr, CCU = Pro, GAG = Glu, GAG = Glu, AAG = Lys.
3. Protein: Met-Val-His-Leu-Thr-Pro-Glu-Glu-Lys…, or MVHLTPEEK in one-letter code.

Mutations

• Silent: a substitution that turns a codon into another for the same amino acid. GTG to GTC in codon 2 still gives valine.
• Missense: the new codon codes for a different amino acid. In sickle-cell disease codon 7 changes from GAG to GTG, so glutamate becomes valine. The hydrophobic valine makes hemoglobin molecules stick together into fibers when oxygen is low, bending red cells into sickles. Some missense changes are harmless; others destroy the protein.
• Nonsense: the codon becomes a stop. Changing base 19 of HBB from G to T turns GAG into TAG, and the protein stops after six amino acids. Mutations like this cause β-thalassemia.
• Frameshift: inserting or deleting a number of bases that is not a multiple of three shifts the reading frame, so every codon after it is different. Deleting base 20 of HBB gives MVHLTPGRSLPLLPCGAR and then a stop: a short, useless protein. Inserting or deleting three bases removes or adds one amino acid without a shift, as in the most common cystic fibrosis mutation, which deletes one phenylalanine.

Using the simulation

In the Protein Synthesis Simulator: Transcription and Translation, things to try:

• Watch β-globin being transcribed, then translated; compare the mRNA in the results with the coding strand.
• Choose Substitution at base 20 with T: the sickle-cell mutation, shown as missense E7V.
• Base 19 to T: nonsense. Base 6 to C: silent.
• Deletion at base 20: frameshift, and the hydropathy chart scrambles after codon 7.
• The short example has its start codon after a few bases: the ribosome skips them, and stops at TGA.
• Type a sequence with no ATG: no protein is made.

What the model assumes

• The standard genetic code, read from the first ATG in the sequence, in that frame, until the first stop codon or the end of the sequence.
• A coding sequence with no introns: what you type is treated as the mRNA's sequence, with no promoter, splicing, cap or tail.
• One polymerase and one ribosome at a time, at fixed speeds; real genes are transcribed by many polymerases and each mRNA is read by many ribosomes at once (a polysome).
• Masses from average residue masses plus one water, with no modifications; methionine is kept at the start.
• Hydropathy from the Kyte–Doolittle scale, averaged over windows of five residues.

Edge cases

• No start codon: no protein. A mutation in the ATG itself loses the start; the simulation then translates from the next ATG, which may be in another frame.
• No stop codon: the ribosome runs off the end of the sequence. In cells such "non-stop" mRNAs are recognized and destroyed.
• Mutations before the start or after the stop leave the protein unchanged, though in real genes they can still affect how much is made.
• A substitution to the same base changes nothing.

Where the model stops being right

• Silent is not always harmless. Synonymous changes can alter splicing signals, mRNA folding or how fast a codon is read, and so change how much protein is made or how it folds.
• Nonsense-mediated decay. In eukaryotes, an mRNA with a premature stop is usually destroyed before much truncated protein is made.
• Start codons in context. Ribosomes in eukaryotes scan from the cap and prefer an AUG in a good context (the Kozak sequence, like GCCACCAUGG); bacteria use a Shine–Dalgarno sequence just upstream. The first ATG is not always the one used.
• After translation. The starting methionine is often removed, which is why the sickle mutation is called Glu6Val; proteins are cut, folded with help from chaperones, and decorated with sugars and phosphates.
• Other codes. Mitochondria and some microbes read a few codons differently; in human mitochondria UGA codes for tryptophan.

Related tools

Translate any sequence in all six frames with Translate DNA to Protein, find open reading frames with the ORF Finder, make point mutations with Mutate DNA, and weigh the result with Protein Molecular Weight. How to Translate DNA to Protein works through the code by hand, and the DNA and RNA Folding Viewer shows the molecule itself.