Termination of translation. The three codons UAG (Amber), UAA (Ochre), and UGA (Opal) are usually translation termination signals. Three proteins, called release factors, are also required for termination. Release Factor 1 is required for termination at UAG and UAA codons, release factor 2 is required for termination at UAA and UGA codons, and release factor 3 accelerates the release of ribosomes from the mRNA. Cells contain about 1 molecule of release factor per 5 ribosomes.
Nonsense suppressors. Nonsense suppressors are produced by base substitution mutations in the DNA corresponding to the anticodon of a tRNA that cause the anticodon to pair with one of the terminarion (or "nonsense") codons, UAG (Amber), UAA (Ochre), or UGA (Opal). Examples of nonsense suppressors produced by single base substitutions in E. coli are shown in the following table. Note that because of the wobble rules amber suppressing tRNAs can only read UAG codons, but ochre-suppressing tRNAs can read both UAA and UAG codons.
|Suppressor||Type||Anticodon change||tRNA||Gene||Map position||Efficiency|
|supE||Amber||CUG --> CUA||tRNAGln||glnV||16 min||0.8-20%|
|supP||Amber||CAA --> CUA||tRNALeu||leuX||97 min||30-100%|
|supD||Amber||CGA --> CUA||tRNASer||serU||43 min||6-54%|
|supU||Amber||CCA --> CUA||tRNATrp||trpT||84 min|
|supF||Amber||GUA --> CUA||tRNATyr||tyrT||27 min||11-100%|
|supZ||Amber||GUA --> CUA||tRNATyr||tyrU||90 min|
|supB||Ochre||UUG --> UUA||tRNAGln||glnU||16 min|
|supL||Ochre||UUU --> UUA||tRNALys||lysT||17 min|
|supN||Ochre||UUU --> UUA||tRNALys||lysV||52 min|
|supC||Ochre||GUA --> UUA||tRNATyr||tyrT||27 min|
|supM||Ochre||GUA --> UUA||tRNATyr||tyrU||90 min|
|glyT||Opal||UCC --> UCA||tRNAGly||glyT||90 min|
|trpT||Opal||CCA --> UCA||tRNATrp||trpT||84 min||0.1-30%|
| The suppression efficiency varies for different sites because the mRNA sequence following the nonsense codon influences how well the suppressor tRNA works. Modified from Miller (1992).|
| Note that this suppressor tRNA can still recognize the wild-type Trp codon as well as the UGA codon.|
Translation termination in sup mutants. Two possible events can occur when a ribosome encounters a nonsense codon in a strain with a nonsense suppressor: (1) termination of peptide elongation can occur if the appropriate release factors associate with the ribosome, or (2) an amino acid can be inserted into the growing peptide chain if the suppressor tRNA associates with the ribosome. The efficiency of suppression depends upon how well the suppressor tRNA is charged with the appropriate amino acid, the concentration of the suppressor tRNA in the cell, and the "context" of the nonsense codon in the mRNA -- especially the base on the 3' side of the codon (the reasons for the context effect are still poorly understood; see Bossi, 1985). In fact, UGA is misread by tRNATrp about 1-3% of the time even in wild-type cells. Although suppressor tRNAs are often inefficient, the amount of protein produced is often sufficient to repair the mutant phenotype.
How do normal proteins terminate in cells with nonsense suppressors? A common explanation given for this problem is that there may be tandem nonsense codons at the end of most genes. However, now that we know the DNA sequence of multiple genomes, it is apparent that this explanation is inadequate -- although some genes end with two nonsense codons, many genes end with a single nonsense codon. An alternative explanation relies on the observation that ochre codons are the most common termination signal used at the end of genes. This idea is supported by the observation that although all known ochre suppressors are inefficient, cells containing ochre suppressors are quite sick (possibly because many translation of many genes fails to terminate properly).