Chapter 25 Using the Genetic Code

25.2 Related Codons Represent Chemically Similar Amino Acids Sixty-one of the sixty-four possible triplets code for twenty amino acids. Three codons (stop codons) do not represent amino acids and cause termination. FIGURE 01: The genetic code is triplet

25.2 Related Codons Represent Chemically Similar Amino Acids The genetic code was frozen at an early stage of evolution and is universal. Most amino acids are represented by more than one codon. FIGURE 02: Amino acids have 1-6 codons each

25.2 Related Codons Represent Chemically Similar Amino Acids The multiple codons for an amino acid are synonymous and usually related. third-base degeneracy – The lesser effect on codon meaning of the nucleotide present in the third (3′) codon position. Chemically similar amino acids often have related codons, minimizing the effects of mutation.

25.3 Codon–Anticodon Recognition Involves Wobbling Multiple codons that represent the same amino acid most often differ at the third base position (the wobble hypothesis). FIGURE 03: Third bases have the least influence on codon meanings

25.3 Codon–Anticodon Recognition Involves Wobbling The wobble in pairing between the first base of the anticodon and the third base of the codon results from looser monitoring of the pairing by rRNA nucleotides in the ribosomal A site. FIGURE 04: Wobble in base pairing allows G-U pairs to form

FIGURE 05: Codon–anticodon pairing involves wobbling at the third position

25.4 tRNAs Are Processed from Longer Precursors A mature tRNA is generated by processing a precursor. The 5′ end is generated by cleavage by the endonuclease RNAase P. The 3′ end is generated by multiple endonucleolytic and exonucleolytic cleavages, followed by addition of the common terminal trinucleotide CCA. FIGURE 06: Both ends of tRNA are generated by processing

25.5 tRNA Contains Modified Bases tRNAs contain over 90 modified bases. Modification usually involves direct alteration of the primary bases in tRNA, but there are some exceptions in which a base is removed and replaced by another base. FIGURE 07: Base modifications in tRNA vary in complexity.

25.5 tRNA Contains Modified Bases Known functions of modified bases are to confer increased stability to tRNAs, and to modulate their recognition by proteins and other RNAs in the translational apparatus.

25.6 Modified Bases Affect Anticodon–Codon Pairing Modifications in the anticodon affect the pattern of wobble pairing and therefore are important in determining tRNA specificity. FIGURE 09: Modification to 2-thiouridine restricts pairing to A FIGURE 08: Inosine pairs with three bases

25.7 There Are Sporadic Alterations of the Universal Code Changes in the universal genetic code have occurred in some species. These changes are more common in mitochondrial genomes, where a phylogenetic tree can be constructed for the changes. FIGURE 11: Changes in the genetic code in mitochondria can be traced in phylogeny

25.7 There Are Sporadic Alterations of the Universal Code In nuclear genomes, the changes usually affect only termination codons. FIGURE 10: Changes in the genetic code usually involve Stop/None signals

25.8 Novel Amino Acids Can Be Inserted at Certain Stop Codons The insertion of selenocysteine at some UGA codons requires the action of an unusual tRNA in combination with several proteins. The unusual amino acid pyrrolysine can be inserted at certain UAG codons. The UGA codon specifies both selenocysteine and cysteine in the ciliate Euplotes crassus. FIGURE 12: SelB is specific for Seleno-Cys-tRNA

25.9 tRNAs Are Selectively Paired with Amino Acids by Aminoacyl-tRNA Synthetases Aminoacyl-tRNA synthetases are a family of enzymes that attach amino acid to tRNA, generating aminoacyl-tRNA in a two-step reaction that uses energy from ATP. Each tRNA synthetase aminoacylates all the tRNAs in an isoaccepting (or cognate) group, representing a particular amino acid.

25.9 tRNAs Are Selectively Paired with Amino Acids by Aminoacyl-tRNA Synthetases Recognition of tRNA by tRNA synthetases is based on a particular set of nucleotides, the tRNA “identity set,” that often are concentrated in the acceptor stem and anticodon loop regions of the molecule. FIGURE 13: An aminoacyl-tRNA synthetase charges tRNA with an amino acid

25.10 Aminoacyl-tRNA Synthetases Fall into Two Families Aminoacyl-tRNA synthetases are divided into class I and class II families based on mutually exclusive sets of sequence motifs and structural domains. FIGURE 16: Class I (Glu-tRNA synthetase) & Class II (Asp-tRNA synthetase) FIGURE 14: Separation of tRNA synthetases into two classes

25.11 Synthetases Use Proofreading to Improve Accuracy Specificity of amino acid-tRNA pairing is controlled by proofreading reactions that hydrolyze incorrectly formed aminoacyl adenylates and aminoacyl-tRNAs. kinetic proofreading – A proofreading mechanism that depends on incorrect events proceeding more slowly than correct events, so that incorrect events are reversed before a subunit is added to a polymeric chain.

FIGURE 17: Kinetic proofreading reduces errors

25.11 Synthetases Use Proofreading to Improve Accuracy chemical proofreading – A proofreading mechanism in which the correction event occurs after the addition of an incorrect subunit to a polymeric chain, by means of reversing the addition reaction. FIGURE 18: Synthetases use chemical proofreading

25.12 Suppressor tRNAs Have Mutated Anticodons That Read New Codons A suppressor tRNA typically has a mutation in the anticodon that changes the codons to which it responds.

25.12 Suppressor tRNAs Have Mutated Anticodons That Read New Codons When the new anticodon corresponds to a termination codon, an amino acid is inserted and the polypeptide chain is extended beyond the termination codon. This results in nonsense suppression at a site of nonsense mutation, or in readthrough at a natural termination codon.

FIGURE 21: Nonsense mutations can be suppressed by a tRNA with a mutant anticodon

25.12 Suppressor tRNAs Have Mutated Anticodons That Read New Codons Missense suppression occurs when the tRNA recognizes a different codon from usual, so that one amino acid is substituted for another. FIGURE 22: Missense suppressors compete with wild type

25.13 There Are Nonsense Suppressors for Each Termination Codon Each type of nonsense codon is suppressed by a tRNA with a mutated anticodon. Some rare suppressor tRNAs have mutations in other parts of the molecule. FIGURE 23: Suppressors have anticodon mutations

25.14 Suppressors May Compete with Wild-Type Reading of the Code Suppressor tRNAs compete with wild-type tRNAs that have the same anticodon to read the corresponding codon(s). Efficient suppression is deleterious because it results in readthrough past normal termination codons. The UGA codon is leaky and is misread by Trp-tRNA at 1% to 3% frequency.

FIGURE 24: Nonsense suppressors read through natural termination codons

25.15 The Ribosome Influences the Accuracy of Translation The structure of the 16S rRNA at the P and A sites of the ribosome influences the accuracy of translation. FIGURE 25: The ribosome selects aminoacyl-tRNAs

25.16 Frameshifting Occurs at Slippery Sequences The reading frame may be influenced by the sequence of mRNA and the ribosomal environment. recoding – Events that occur when the meaning of a codon or series of codons is changed from that predicted by the genetic code. It may involve altered interactions between aminoacyl-tRNA and mRNA that are influenced by the ribosome.

25.16 Frameshifting Occurs at Slippery Sequences Slippery sequences allow a tRNA to shift by one base after it has paired with its anticodon, thereby changing the reading frame. Translation of some genes depends upon the regular occurrence of programmed frameshifting. FIGURE 26: A tRNA that slips one base in pairing with a codon causes a frameshift that

25.16 Frameshifting Occurs at Slippery Sequences FIGURE 27: Bypassing skips between identical codons

FIGURE 28: Frameshifting controls translation 25.17 Other Recoding Events: Translational Bypassing and the tmRNA Mechanism to Free Stalled Ribosomes Bypassing involves the capacity of the ribosome to stop translation, release from mRNA, and resume translation some 50 nucleotides downstream. FIGURE 29: In bypass mode, a ribosome with its P site occupied can stop translation FIGURE 28: Frameshifting controls translation

25.17 Other Recoding Events: Translational Bypassing and the tmRNA Mechanism to Free Stalled Ribosomes Ribosomes that are stalled on mRNA after partial synthesis of a protein may be freed by the action of tmRNA, a unique RNA that incorporates features of both tRNA and mRNA.