A gene sitting in DNA does nothing by itself. Your liver cells and nerve cells carry identical DNA, yet one makes the blood protein albumin and the other makes neurotransmitter receptors, because each reads a different set of genes. Reading a gene happens in two stages. First the cell copies the gene's sequence into a disposable RNA message (transcription); then ribosomes decode that message, three bases at a time, into a chain of amino acids (translation). Francis Crick called this one-way flow of information, DNA RNA protein, the central dogma (1958). Why have a middle step at all? DNA is the master copy and stays protected in the nucleus, while many RNA copies can be made, sent out to ribosomes, and destroyed when no longer needed, which lets the cell control how much of each protein it makes. (Some viruses, such as HIV, break the one-way rule: their enzyme reverse transcriptase copies RNA back into DNA.) Following a gene from DNA to protein is the skill behind understanding mutations, genetic disease and biotechnology.
RNA and its three main jobs
- Sugar: deoxyribose (no –OH on the 2' carbon)
- Bases: A, T, G, C
- Usually a double-stranded helix
- Very long: a whole chromosome is one molecule
- Chemically stable; stays in the nucleus in eukaryotes
- Job: long-term storage of genetic information
- Sugar: ribose (–OH on the 2' carbon)
- Bases: A, U, G, C (uracil replaces thymine and pairs with A)
- Usually single-stranded, but can fold back on itself
- Short: usually a copy of one gene or less
- Less stable (the 2'-OH makes it easier to break); many kinds leave the nucleus
- Job: carries, decodes and helps carry out the genetic message
| RNA | Structure | Job |
|---|---|---|
| mRNA (messenger) | Single strand copied from one gene, with untranslated sequences at each end. | Carries the gene's code from the DNA to a ribosome as a series of three-base codons. |
| tRNA (transfer) | About 70–90 nucleotides folded into a cloverleaf (an L-shape in 3-D). One loop holds the three-base anticodon; the 3' end carries an amino acid. | Brings the correct amino acid to the ribosome by pairing its anticodon with the mRNA codon. Enzymes called aminoacyl-tRNA synthetases attach the right amino acid to each tRNA. |
| rRNA (ribosomal) | Combines with proteins to form the small and large ribosomal subunits. | Forms the ribosome's core and catalyzes peptide bond formation, so the ribosome is an RNA enzyme (a ribozyme). |
Transcription: DNA to mRNA
- Initiation. RNA polymerase binds a promoter, a DNA sequence just upstream of the gene that marks where to start and which strand to read. In bacteria a protein subunit called sigma factor recognizes the promoter. In eukaryotes, proteins called transcription factors first bind the promoter, often at the TATA box (a T- and A-rich sequence about 25–30 base pairs upstream of the start), and then recruit RNA polymerase II. RNA polymerase unwinds the DNA itself and, unlike DNA polymerase, needs no primer.
- Elongation. Only one DNA strand, the template strand (antisense strand), is read. RNA polymerase moves along it 3'→5' and joins complementary RNA nucleotides, so the mRNA grows 5'→3' (new nucleotides always go on the 3' end, just as in replication). An A in the template gives a U in the RNA. The other strand, the coding strand (sense strand), is not read, but it has the same sequence as the mRNA with T in place of U. The DNA rewinds behind the polymerase.
- Termination. In bacteria, RNA polymerase transcribes a terminator sequence; the new RNA folds into a hairpin loop that pulls it off the DNA. In eukaryotes, transcription runs past a polyadenylation signal (AAUAAA in the RNA), and the RNA is cut free a short distance after it.
In eukaryotes the first transcript, called pre-mRNA, is modified in the nucleus before it is exported. A 5' cap, a modified guanine nucleotide, is added to the 5' end; it protects the mRNA from being digested and helps the ribosome attach. A poly-A tail of about 50–250 adenine nucleotides is added to the 3' end; it slows breakdown and helps export the mRNA from the nucleus. Most important, most eukaryotic genes are interrupted by non-coding introns (intervening sequences) between the coding exons (expressed sequences). A complex of RNA and proteins called a spliceosome cuts the introns out and joins the exons together, a process called splicing. Introns often make up most of a gene's length. Through alternative splicing, different cells join different combinations of exons from the same pre-mRNA, which is how roughly 20 000 human genes can produce many more different proteins. Bacterial genes have no introns, and bacterial mRNA gets no cap or tail.
The genetic code
- Triplet: each codon is three nucleotides. A two-letter code would give only combinations, too few for 20 amino acids; three letters give .
- Start and stop: AUG codes for methionine (Met) and is the start codon, which sets the reading frame. UAA, UAG and UGA are stop codons and code for no amino acid. That leaves 61 codons for 20 amino acids.
- Redundant (degenerate): most amino acids have more than one codon. Leucine, serine and arginine each have six; only methionine (AUG) and tryptophan (UGG) have one. Codons for the same amino acid usually differ only in the third base, so many third-base mutations change nothing.
- Unambiguous: each codon specifies only one amino acid. GGU always means glycine.
- Non-overlapping, with no gaps: codons are read one after another from the start codon, so inserting or deleting one base shifts every codon after it.
- Nearly universal: the same codons mean the same amino acids in bacteria, plants and humans (with small exceptions in mitochondria and a few microbes). That is why a human gene can be expressed in bacteria, and it is strong evidence that all life shares a common ancestor.
Translation: mRNA to polypeptide
- Initiation: the small ribosomal subunit binds the mRNA (in eukaryotes at the 5' cap, then it scans to the first AUG; in bacteria at a ribosome-binding sequence just before the AUG). An initiator tRNA with anticodon 3'-UAC-5', carrying methionine, pairs with the AUG. The large subunit then joins, with the initiator tRNA in the P site (peptidyl site).
- Codon recognition: a tRNA whose anticodon is complementary to the codon in the empty A site (aminoacyl site) enters with its amino acid. The ribosome checks the codon–anticodon pairing, not the amino acid.
- Peptide bond formation: rRNA in the large subunit joins the chain on the P-site tRNA to the amino acid on the A-site tRNA with a peptide bond. The whole growing chain is now held by the A-site tRNA.
- Translocation: the ribosome moves one codon (three nucleotides) toward the 3' end of the mRNA. The tRNA holding the chain shifts into the P site, the empty tRNA shifts to the E site (exit site) and leaves, and the A site is open for the next codon. Steps 2 to 4 repeat, powered by GTP.
- Termination: when a stop codon (UAA, UAG or UGA) reaches the A site, no tRNA matches it. A protein called a release factor binds instead, the bond holding the polypeptide to the last tRNA is broken, the chain is released, and the subunits separate.
The ribosome reads mRNA 5'→3', and the polypeptide grows from its amino (N) end to its carboxyl (C) end. Several ribosomes can translate the same mRNA at once, each a little further along; the cluster is called a polysome (polyribosome), and it multiplies the protein output of a single message. The new chain then folds, often with help from chaperone proteins, and may be modified before it works. In prokaryotes there is no nuclear envelope, so ribosomes attach to the mRNA while it is still being transcribed: transcription and translation are coupled. In eukaryotes the two are separated in space and time: transcription and processing happen in the nucleus, and translation happens in the cytoplasm, on free ribosomes or on ribosomes bound to the rough endoplasmic reticulum (for proteins that will be secreted or placed in membranes).