Gene Expression II: Translation at the Ribosome

The world's most important factory

Every protein in your body, from the keratin in your hair to the antibodies in your blood, is assembled by ribosomes. A growing cell can contain millions of them. Each one reads an mRNA three bases at a time and links the matching amino acids together in the right order. In bacteria a ribosome adds roughly 15 to 20 amino acids per second.

In the last two lessons you made a mature mRNA and learned the genetic code. Now you will see the machine that actually reads the code. You will also investigate it with a model (expectation D2.4).

The adaptor: transfer RNA

Codons cannot recognize amino acids directly. Crick predicted an "adaptor" molecule, and it turned out to be transfer RNA (tRNA): a single strand of about 76 to 90 nucleotides that folds into a cloverleaf shape (L-shaped in three dimensions).

• At its 3′ end (always ending in CCA), it carries one specific amino acid.
• In a loop at the other end it has the anticodon, three bases that pair with a codon on the mRNA.

Pairing is antiparallel: the codon 5′-AUG-3′ pairs with the anticodon 3′-UAC-5′. (If an anticodon is written 5′→3′, as is common, it reads CAU.)

Charging tRNAs. An enzyme family called aminoacyl-tRNA synthetases attaches each amino acid to the right tRNAs, using energy from ATP. There is one synthetase for each of the 20 amino acids, and each one recognizes both its amino acid and its tRNAs. These enzymes are where the genetic code is really "enforced": if a synthetase put the wrong amino acid on a tRNA, the ribosome would insert it without noticing.

Wobble. Cells have fewer kinds of tRNA than the 61 sense codons. The third base of a codon and the first base of the anticodon can pair loosely (for example, G in the anticodon can pair with U or C), so one tRNA can read several codons for the same amino acid.

The ribosome

A ribosome is made of ribosomal RNA (rRNA) and dozens of proteins, in two subunits:

• Prokaryotes: small 30S + large 50S = 70S ribosome.
• Eukaryotes: small 40S + large 60S = 80S ribosome (in the cytosol and on the rough ER).

The "S" is a Svedberg unit, a measure of how fast a particle settles in a centrifuge, which depends on shape as well as mass. That is why 30 + 50 does not equal 70.

The large subunit has three sites for tRNAs: the A site (aminoacyl: the new tRNA arrives), the P site (peptidyl: holds the growing chain) and the E site (exit). The small subunit holds the mRNA and checks codon-anticodon pairing. The bond-forming activity, peptidyl transferase, belongs to the rRNA of the large subunit, not to a protein. The ribosome is a ribozyme, an RNA enzyme. Venkatraman Ramakrishnan, Thomas Steitz and Ada Yonath shared the 2009 Nobel Prize in Chemistry for mapping the ribosome's atomic structure.

Stage 1: initiation

1. The small subunit binds the mRNA near its 5′ end.

• In bacteria, a short sequence a few bases upstream of the start codon, the Shine-Dalgarno sequence, pairs with rRNA in the small subunit and lines up the start codon.
• In eukaryotes, the small subunit binds the 5′ cap and scans along the mRNA until it finds the first AUG (usually).

1. The initiator tRNA, carrying methionine (a modified form, formyl-methionine, in bacteria), pairs with the AUG start codon. It is the only tRNA that goes directly into the P site.
2. The large subunit joins. Proteins called initiation factors help, and energy comes from GTP.

Stage 2: elongation

This three-step cycle repeats for every codon:

1. Codon recognition. An aminoacyl-tRNA whose anticodon matches the codon in the A site binds there. (Proteins called elongation factors bring it in, using GTP.)
2. Peptide bond formation. Peptidyl transferase moves the growing chain from the tRNA in the P site and joins it with a peptide bond to the amino acid on the tRNA in the A site. Now the A-site tRNA holds the chain.
3. Translocation. The ribosome moves one codon (three bases) toward the 3′ end of the mRNA, using GTP. The tRNA with the chain shifts to the P site, the empty tRNA shifts to the E site and leaves, and the A site is open for the next codon.

The mRNA is read 5′→3′, and the polypeptide is built from its N-terminus (amino end, the first amino acid) to its C-terminus (carboxyl end).

Stage 3: termination

When a stop codon (UAA, UAG or UGA) enters the A site, no tRNA matches it. A protein called a release factor binds instead. It triggers hydrolysis of the bond between the polypeptide and the last tRNA, the polypeptide is released, and the subunits separate, ready to be reused.

After translation

• Polysomes. Many ribosomes can read one mRNA at the same time, each a little farther along, so many copies of the protein are made quickly.
• Where proteins go. Proteins for the cytosol are made on free ribosomes. Proteins that will be secreted or placed in membranes begin with a signal sequence of amino acids; it directs the ribosome to the rough endoplasmic reticulum, and the protein travels on through the Golgi apparatus (you studied these organelles in the Biochemistry unit).
• Finishing touches. A new polypeptide must fold into its 3-D shape, often helped by chaperone proteins. Many proteins are also modified: the hormone insulin, for example, is made as one longer chain, proinsulin, which is then cut to give the active hormone; other proteins get sugars, phosphates or other groups added.

The energy cost

For each amino acid added, the cell spends about four high-energy phosphate bonds: two to charge the tRNA (ATP → AMP + 2 phosphates) and two GTPs (one to bring in the tRNA, one for translocation). Protein synthesis is one of the most energy-expensive things a cell does.

Medical connection: antibiotics that target ribosomes

Because bacterial 70S ribosomes differ from our 80S ribosomes, several antibiotics can block bacterial protein synthesis while doing much less to human cells:

• Tetracyclines block the A site of the 30S subunit, so aminoacyl-tRNAs cannot bind.
• Streptomycin binds the 30S subunit and causes misreading of codons.
• Erythromycin binds the 50S subunit and blocks the tunnel through which the new chain leaves.
• Chloramphenicol blocks peptidyl transferase.

Mitochondria have their own ribosomes that resemble bacterial ones (a clue to their bacterial origin), which explains some side effects of these drugs.