11 The Genetic Code & Translation To understand how mRNA is At the heart of the central dogma is the concept that information in the Goal translated into protein. form of the four-letter alphabet (A, G, C and T) of the genetic material is translated into the 20-letter (amino acids) alphabet of proteins. As we Objectives have seen, the intermediary between the genetic material, DNA, and the After this chapter, you should be able to translation machinery, the ribosome, is the messenger RNA or mRNA. The mRNA is copied from the DNA in a process called transcription (Chapter • describe the principal features of the 10) and is then decoded on the ribosome in a process called translation, genetic code. where it directs the ordered polymerization of amino acids into polypeptide • explain how tRNAs mediate chains. Here we focus on the nature and logic of the genetic code, the RNA information transfer and do so adaptors that decipher the genetic code, the workings of the molecular accurately. machine that translates mRNA into protein and does so with high accuracy, • describe the peptide bond cycle and and the chemistry of the formation of the peptide bond. how it achieves accuracy. • describe the ribosome cycle and how open reading frames are set. The four-letter alphabet of the genetic material is read in units of three How many bases are required to specify an amino acid? Because nucleic acids have only four bases and proteins are composed of twenty amino acids, the coding unit or codon for each amino acid must consist of more than one base. Even two bases would not be enough. If codons consisted of two bases, then only 16 (or 42) different codons would be possible, and there would insufficient codons to specify 20 amino acids. However, if codons are composed of three bases, then 64 (or 43) codons are possible, more than enough for the amino acid alphabet. Therefore, the minimum number of bases needed to specify 20 different amino acids is three. Indeed, the genetic code is a triplet code. Chapter 11 The Genetic Code & Translation 2 Second position Figure 1 Each codon corresponds to a particular amino acid U C A G UUU UCU UAU UGU U Each codon is written from 5’ to 3’. The Phe Tyr Cys start codon (AUG) is shown in green. Stop UUC UCC UAC UGC C U Ser codons (UAA, UAG, and UGA) are shown UUA UCA UAA UGA Stop A in red. Leu Stop UUG UCG UAG UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg position (3’) Third CUA CCA CAA CGA A Gln CUG CCG CAG CGG G AUU ACU AAU AGU U Asn Ser First Position (5’) Position First AUC Ile ACC AAC AGC C A Thr AUA ACA AAA AGA A Lys Arg AUG Met ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G Of the 64 possible codons in the genetic code, 61 specify amino acids, indicating that many amino acids are encoded by more than one codon (Figure 1). Thus, the code isdegenerate in the sense that some amino acids are specified by more than one synonymous codon. At one extreme, leucine, serine, and arginine are each specified by six synonymous codons. At the other extreme, methionine and tryptophan have unique codons (AUG and UGG, respectively). The methionine codon AUG has an additional function as a start codon: it signals the beginning of a coding sequence in the mRNA. The remaining three (of the 64) triplets—UAA, UGA, and UAG—do not specify any amino acids. Instead, these triplets are stop codons that signal the end of the coding sequence for a messenger RNA. Thus, coding sequences have two kinds of punctuation marks: an AUG at the beginning that marks the start and one (or more successive) stop codons at the end. We will return to start and stop codons near the end of the chapter, including the question of how AUG can serve both as a methionine codon internal to a coding sequence and as the start signal at the beginning. The entire genetic code, which is sometimes called the “Rosetta Stone of Life” (because it deciphers codons), is shown in its entirety in Figure 1. Notice that the left-hand vertical column indicates the first (5’) position in a codon, the horizontal bar across the top indicates the second position, and the right-hand vertical column indicates the third (3’) position. Start and stop codons are highlighted in green and red, respectively. Finally, we return to the 5’-to-3’ directionality of polynucleotides. Codons have a 5’-to-3’ orientation with respect to the directionality of the RNA Chapter 11 The Genetic Code & Translation 3 transcript in which they are embedded. Thus, for example, the CGA codon for arginine has the orientation 5’-CGA-3’. This is in keeping with the three foundational rules of directionality introduced in Chapter 8: polynucleotide synthesis proceeds in a 5’-to-3’ direction (rule 1), polypeptide synthesis proceeds in an NH2-terminal-to-COOH-terminal direction (rule 2), and information for the order of amino acids in the mRNA is specified sequentially in a 5’-to-3’ direction (rule 3). Thus, codons are lined up in the same 5’-to-3’ orientation as the direction of translation. Transfer RNAs are adaptors between codons and amino acids How are the 61 codons that specify amino acids deciphered such that each directs the incorporation of the appropriate, cognate amino acid? The answer is that codons are recognized by adaptor molecules known as transfer RNAs or tRNAs. In contrast to mRNAs, tRNAs are non-protein- coding RNAs that directly act as adaptors through their tertiary structure, as we will explain. (A second example of non-coding RNAs is the RNA components of the ribosome, as we will also discuss.) Each tRNA recognizes the codon for a particular amino acid. Recognition is mediated by base pairing between the codon and a corresponding anti-codon in the tRNA molecule, which align in an anti-parallel orientation. Covalently attached to each tRNA at its 3’ terminus is its cognate amino acid, that is, the amino (A) O acid that corresponds to that specified by the codon. Thus, the amino acid N NH aspartic acid is covalently attached to a tRNA whose anti-codon pairs with the aspartic acid codon 5’-GAC-3’. N N Some tRNAs recognize more than one synonymous codon. This is possible Inosine due to a phenomenon known as wobble, which takes advantage of the fact that synonymous codons often differ from each other at the third (3’) (B) H position. Part of the explanation for wobble is that the 5’ base in the anti- N O H N codon is not as spatially restricted as the other two, allowing it to “wobble” and form hydrogen bonds with bases other than its cognate base at the 3’ N I N H N C position of the codon. Also, some tRNAs have an unusual base, inosine N N (Figure 2A), at the 5’ (wobble) position of the anti-codon; inosine is able O to pair with A, U or C at the 3’ position of the codon (Figure 2B). Thus, the base-pair rules that form the basis for double-helical structures are not O strictly adhered to in the special case of codon/anti-codon interactions. N O H N U tRNAs are approximately 80 nucleotides in length. They contain regions N N of self-complementarity that enable them to fold back on themselves in a I N H O N characteristic cloverleaf-like pattern of loops and short stretches of double helix (analogous to secondary structure in proteins). The cloverleaf, in turn, folds into a precise three-dimensional structure (analogous to the tertiary H structure of proteins) that resembles the capital letter “L” (an upside-down N N N O H L in Figure 3). A key feature of tRNAs is that the anti-codon and the site of N A N attachment of the amino acid are at opposite ends of the L-shaped molecule. I N H N N N The anti-codon is displayed in a loop at one end, and the amino acid is attached to the 3’ terminus at the other end. Notice that the 5’ and 3’ termini Figure 2 Inosine can form base are both near the same end of the molecule but that the 3’ end protrudes as pairs with A, U, or C a short stretch of single-stranded RNA beyond the 5’ terminus. Chapter 11 The Genetic Code & Translation 4 (A) (B) amino acid attachment point 3’ 5’ 3’ 5’ amino acid attachment point anti-codon anti-codon Figure 3 tRNAs exhibit secondary and tertiary structure (A) Shown is a cartoon representing the two-dimensional, cloverleaf-like secondary structure of a generic tRNA. Each circle represents an individual nucleotide, with the nucleotides of the anti-codon shown in blue. Black lines indicate base pairing within the strand. (B) Shown is the X-ray crystal structure of a tRNA molecule. The tRNA folds into an L-shaped structure, with the anti-codon at one end and the 3’ hydroxyl, which is where the amino acid becomes attached, at the other end. All tRNAs have this overall folded structure, even though their specific nucleotide sequences vary. Shown is a tRNA for phenylalanine; as such, it is known as tRNAPhe.