The Ribosome and Peptidyl Transfer
Ribozyme Catalysis, Aminoacyl-tRNA Kinetic Proofreading, and Polysome Assembly Lines
George Palade discovered ribosomes in 1955 as dense 20-nanometer granules carpeting the endoplasmic reticulum in electron micrographs. In a rapidly dividing Escherichia coli cell, 50,000 to 70,000 ribosomes make up nearly 30 percent of the bacterium's total dry weight. Working continuously, those particles forge two million protein molecules every twenty minutes.
When Thomas Steitz and Peter Moore solved the $2.4\text{-\AA}$ X-ray crystal structure of the haloarchaeal $50\text{S}$ large ribosomal subunit in 2000, they uncovered a striking evolutionary fact. Zero protein amino acid side chains sit within $18\text{ \AA}$ of the active site where peptide bonds form. The ribosome is an ancient RNA ribozyme braced by external proteins.
Without protein histidine or cysteine side chains at the active site, how does $23\text{S}$ ribosomal RNA forge 15 to 20 peptide bonds per second? Structural studies by Marina Rodnina and Scott Strobel showed that the rRNA pocket aligns its substrates entropically. It routes a proton shuttle directly through the $2'\text{-OH}$ group of the P-site tRNA's terminal adenosine-76.
Venki Ramakrishnan solved the $3.0\text{-\AA}$ structure of the $30\text{S}$ small subunit in 2000, revealing how the decoding center reads genetic letters. Two conserved $16\text{S}$ rRNA adenines, A1492 and A1493, flip out of helix 44 like molecular calipers. They probe the minor groove of the first two codon-anticodon pairs to enforce strict Watson-Crick geometry.
Equilibrium binding energy between a three-base codon and anticodon can distinguish a single-letter mismatch by only about 100-to-1. John Hopfield in 1974 and Jacques Ninio in 1975 showed that the ribosome reaches a $10^{-4}$ error rate via kinetic proofreading. Elongation factor EF-Tu hydrolyzes GTP to insert an irreversible delay that tests tRNA binding twice.
The ribosome itself never checks which amino acid is attached to an incoming tRNA; it trusts the twenty aminoacyl-tRNA synthetase enzymes. As Linus Pauling predicted and Alan Fersht proved in 1977, isoleucyl-tRNA synthetase rejects the one-methylene-smaller valine by 3,000-to-1. A separate double-sieve editing pocket hydrolyzes mischarged valyl-tRNA before it leaves the enzyme.
While A1492 and A1493 enforce rigid geometry on the first two letters of a codon, the third position sits in a roomier slot predicted by Francis Crick's 1966 wobble hypothesis. Non-standard base pairs like guanine-uracil are tolerated at that third letter. Wobble pairing lets a cell decode all 61 sense codons using only 30 to 45 tRNA species.
After each peptide bond forms, the GTPase factor EF-G catalyzes translocation by ratcheting the $30\text{S}$ subunit 7 degrees relative to the $50\text{S}$ subunit. Single-molecule optical tweezers by Carlos Bustamante measured the power of this pawl. The ribosome pulls messenger RNA forward in three-nucleotide steps ($1.3\text{ nm}$) against opposing loads up to $13\text{ pN}$.
Every growing polypeptide chain threads out of the ribosome through a 10-nanometer-long, $1.5\text{-nm}$-wide exit tunnel carved through the $50\text{S}$ subunit. Rather than acting as a passive pipe, the tunnel's constricted uL4-uL22 ring inspects the passing chain. Specific nascent peptides like SecM jam themselves against the tunnel wall, stalling translation to regulate secretion genes.
No tRNA molecule in normal cells recognizes the stop codons UAA, UAG, or UGA. Instead, protein release factors RF1 and RF2 fold into an L-shaped mimic of a tRNA. When their reading heads match a stop codon in the A-site, a conserved GGQ loop reaches into the catalytic center to coordinate a water molecule that clips the protein free.
When a bacterial messenger RNA breaks in half and lacks a stop codon, the ribosome stalls at the torn $3'$ end. Kenneth Keiler and Robert Sauer discovered in 1996 that a chimeric molecule called tmRNA rescues the trapped machine. Entering the A-site like a tRNA, tmRNA swaps in its own reading frame to add an 11-amino-acid destruction tag.
Alexander Rich discovered in 1962 that cells multiply protein output from one mRNA by stringing 10 to 80 ribosomes along the message simultaneously as a polyribosome, spaced every 80 nucleotides. In eukaryotes, initiation factors bridge the $5'$ cap to the $3'$ poly(A) tail in a closed loop. Terminating ribosomes immediately re-enter at the start codon.