Premed · Premed · Genetics
Lecture 11: Translation and the Genetic Code
Genetics
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the properties of the genetic code (triplet, degenerate, non-overlapping, universal with exceptions)
- Use a codon table to determine amino acid sequences from mRNA
- Explain the structure and function of tRNA and ribosomes
- Describe the three stages of translation: initiation, elongation, and termination
- Compare translation in prokaryotes and eukaryotes
- Explain the wobble hypothesis and its implications for codon-anticodon pairing
Lecture Content
I. The Genetic Code
The genetic code specifies the relationship between the nucleotide sequence of mRNA and the amino acid sequence of a protein. It has several important properties. It is a triplet code, meaning each codon consists of three consecutive nucleotides. It is non-overlapping, so codons are read sequentially without sharing nucleotides, and comma-free, meaning codons are read continuously without gaps or separators. The code is degenerate (redundant): with 64 possible codons encoding only 20 amino acids plus 3 stop signals, most amino acids are specified by more than one codon. Leucine, serine, and arginine are the most degenerate with 6 codons each, while methionine and tryptophan have only 1 codon each. Degeneracy is greatest at the third (wobble) position. The code is nearly universal, used by virtually all organisms with minor exceptions in mitochondria (where UGA encodes tryptophan instead of stop), Mycoplasma, and some ciliates. Critically, the code is unambiguous: each codon specifies exactly one amino acid.
The start codon AUG encodes methionine and also serves as the initiation codon (formyl-methionine in prokaryotes). The three stop codons are UAA (ochre), UAG (amber), and UGA (opal/umber); they do not encode amino acids but instead signal translation termination. The reading frame is established by the start codon, and each strand has three possible reading frames.
II. Cracking the Code: Key Experiments
The genetic code was deciphered through a series of ingenious experiments. In 1961, Nirenberg and Matthaei showed that a synthetic poly-U mRNA directed the synthesis of polyphenylalanine, proving that UUU encodes phenylalanine and opening the door to systematic codon assignments. Nirenberg and Leder then developed a triplet binding assay in which specific trinucleotides bound to ribosomes with specific aminoacyl-tRNAs, allowing the assignment of many additional codons. Khorana synthesized defined polynucleotides with repeating sequences to determine the remaining codon assignments. By 1966, all 64 codons had been assigned.
III. The Wobble Hypothesis
Proposed by Francis Crick in 1966, the wobble hypothesis explains the rules governing codon-anticodon pairing at the third position of the codon. The first two positions of the codon pair strictly with the anticodon through standard Watson-Crick base pairing. However, the third position of the codon (corresponding to the 5' end of the anticodon) allows non-standard "wobble" pairing: anticodon U can pair with codon A or G, anticodon G can pair with codon C or U, and anticodon I (inosine, derived from deamination of adenosine) can pair with codon U, C, or A.
The wobble hypothesis explains several important observations: fewer than 61 tRNA species are needed to decode all sense codons (only about 45 tRNAs exist in humans), degeneracy is concentrated at the third codon position, and silent mutations most commonly occur at the third position.
IV. Transfer RNA (tRNA)
Transfer RNA serves as the adaptor molecule that "translates" the nucleotide language of mRNA into the amino acid language of proteins. Each tRNA is approximately 76-90 nucleotides long, adopts a cloverleaf secondary structure, and folds into an L-shaped three-dimensional structure. Key structural features include the acceptor stem at the 3' end, which carries the CCA sequence to which the amino acid is attached at the 3'-OH of the terminal adenosine; the anticodon loop, containing the three-nucleotide anticodon complementary to the mRNA codon; the D loop, containing dihydrouridine and involved in recognition by aminoacyl-tRNA synthetases; the T-psi-C loop, containing ribothymidine and pseudouridine and involved in ribosome binding; and the variable loop, which varies in size among different tRNAs. Transfer RNAs also contain many modified bases, including inosine, pseudouridine, dihydrouridine, and methylated bases.
Aminoacyl-tRNA synthetases (aaRS) are the 20 enzymes that charge each tRNA with its correct amino acid. The charging reaction proceeds in two steps: first, the amino acid is activated by ATP to form aminoacyl-AMP with release of pyrophosphate; then the aminoacyl group is transferred from AMP to the tRNA. Each synthetase recognizes its cognate tRNA through specific identity elements including the anticodon, acceptor stem, and discriminator base. Many synthetases also possess an editing site that hydrolyzes incorrectly charged amino acids, providing an essential proofreading mechanism.
<image>Panel A: The standard genetic code table organized as a 4x4 grid of codon families, with first position on the left, second position on top, and third position on the right, with start (AUG) and stop codons (UAA, UAG, UGA) highlighted. Panel B: tRNA structure showing cloverleaf secondary structure with labeled arms (acceptor stem with 3'-CCA, anticodon loop, D loop, T-psi-C loop, variable loop) and the L-shaped tertiary structure alongside it. Panel C: Wobble base pairing rules diagram showing standard Watson-Crick pairs at positions 1 and 2, and the non-standard wobble pairs at position 3 of the codon (with inosine, G, and U in the anticodon pairing with multiple bases in the codon).</image>
V. Ribosome Structure
Ribosomes are the molecular machines that carry out translation, composed of ribosomal RNA and ribosomal proteins. The prokaryotic ribosome (70S) consists of a small subunit (30S) containing the 16S rRNA and 21 proteins, and a large subunit (50S) containing the 23S rRNA, 5S rRNA, and 31 proteins. The 16S rRNA participates in mRNA binding through the Shine-Dalgarno interaction and in decoding, while the 23S rRNA functions as the peptidyl transferase, making the ribosome a ribozyme in which RNA catalyzes peptide bond formation.
The eukaryotic ribosome (80S) consists of a small subunit (40S) containing the 18S rRNA and approximately 33 proteins, and a large subunit (60S) containing the 28S, 5.8S, and 5S rRNAs along with approximately 49 proteins. All ribosomes contain three tRNA binding sites: the A site (aminoacyl) accepts incoming aminoacyl-tRNA, the P site (peptidyl) holds the tRNA carrying the growing polypeptide chain, and the E site (exit) holds the deacylated tRNA before it exits the ribosome.
VI. Translation: Initiation
Prokaryotic initiation begins when IF3 binds the 30S subunit to prevent premature association with the 50S, and IF1 binds the A site to block it during initiation. The mRNA then binds to the 30S subunit through base pairing between the Shine-Dalgarno sequence (AGGAGG, located approximately 5-10 nucleotides upstream of AUG) and the 16S rRNA. The initiator tRNA carrying formyl-methionine (fMet-tRNA^fMet) binds the P site, guided by IF2, a GTPase. Finally, the 50S subunit joins, the initiation factors are released, GTP is hydrolyzed, and the 70S initiation complex is formed.
Eukaryotic initiation is considerably more complex, involving more than 12 initiation factors. The process begins when eIF2-GTP binds the initiator Met-tRNA to form a ternary complex, which then associates with the 40S subunit along with eIF1, eIF1A, eIF3, and eIF5 to create the 43S pre-initiation complex. Meanwhile, the eIF4F complex (consisting of eIF4E, eIF4A, and eIF4G) binds the 5' cap of the mRNA. eIF4E is the cap-binding protein, eIF4A is an RNA helicase that unwinds secondary structure in the 5' UTR, and eIF4G is a scaffold protein that bridges the cap and the poly(A) tail via PABP. The 43S complex then scans along the 5' UTR in the 5' to 3' direction until it encounters the first AUG in a favorable Kozak sequence context (gcc(A/G)ccAUGG). AUG recognition triggers GTP hydrolysis by eIF2, initiation factors are released, and the 60S subunit joins to form the 80S initiation complex.
VII. Translation: Elongation
Elongation proceeds through three steps that are repeated cyclically. In the first step, aminoacyl-tRNA delivery (decoding), the aminoacyl-tRNA is delivered to the A site as a ternary complex with EF-Tu (in prokaryotes) or eEF1A (in eukaryotes) and GTP. A correct codon-anticodon match triggers GTP hydrolysis, release of EF-Tu/eEF1A, and accommodation of the aminoacyl-tRNA in the A site. Kinetic proofreading at this step rejects incorrect tRNAs. In the second step, peptide bond formation (transpeptidation), the peptidyl transferase activity of the large ribosomal RNA catalyzes the attack of the alpha-amino group of the A-site amino acid on the carbonyl carbon of the P-site peptidyl-tRNA, transferring the peptide chain from the P-site tRNA to the A-site tRNA. In the third step, translocation, EF-G (prokaryotes) or eEF2 (eukaryotes) with GTP drives the ribosome to translocate one codon (3 nucleotides) in the 5' to 3' direction: the A-site tRNA carrying the peptide moves to the P site, the deacylated P-site tRNA moves to the E site and exits, and the A site is left empty and ready for the next aminoacyl-tRNA. The elongation rate is approximately 15-20 amino acids per second in prokaryotes and about 6 amino acids per second in eukaryotes.
VIII. Translation: Termination
Termination occurs when a stop codon (UAA, UAG, or UGA) enters the A site. No aminoacyl-tRNA recognizes stop codons. Instead, release factors bind the A site: in prokaryotes, RF1 recognizes UAA and UAG while RF2 recognizes UAA and UGA; in eukaryotes, eRF1 alone recognizes all three stop codons. The release factors trigger hydrolysis of the bond between the polypeptide and the tRNA in the P site, releasing the completed polypeptide. RF3 (prokaryotes) or eRF3 (eukaryotes) is a GTPase that facilitates release factor dissociation.
Ribosome recycling then separates the ribosome into its subunits. In prokaryotes, the ribosome recycling factor (RRF) along with EF-G separates the 70S ribosome into 30S and 50S subunits. In eukaryotes, ABCE1, an ATPase, promotes subunit dissociation. The newly synthesized polypeptide then undergoes post-translational modifications including folding (assisted by chaperones), cleavage of signal peptides, glycosylation, phosphorylation, and other modifications.
<image>Panel A: Complete diagram of translation elongation showing the three-step cycle: (1) aminoacyl-tRNA-EF-Tu-GTP entering the A site with codon-anticodon recognition, (2) peptide bond formation catalyzed by peptidyl transferase center with the peptide chain transferred to A-site tRNA, (3) EF-G-GTP-driven translocation moving tRNAs from A→P and P→E sites. Panel B: Comparison of prokaryotic and eukaryotic translation initiation: prokaryotic showing Shine-Dalgarno interaction with 16S rRNA and fMet-tRNA placement; eukaryotic showing cap-dependent scanning model with eIF4F, 43S PIC, and Kozak sequence recognition. Panel C: Translation termination diagram showing a stop codon in the A site, release factor binding, peptidyl-tRNA hydrolysis, polypeptide release, and ribosome recycling into subunits.</image>
