Why is it possible for an amino acid to be specified by more than one codon

DNA Structure and the Genetic Code

Humans store genetic information in DNA, a linear polymer made up of four different nucleotides: adenine (A), guanine (G), thymidine (T), and cytosine (C). Nucleotides, also calledbases, are linked together by phosphodiester bonds into a single strand. Nucleotides also have the capability of pairing with each other (A with T and G with C) through hydrogen bonds. Two strands of DNA can pair with each other in a complementary fashion—again, A with T and G with C—to form a double helix. The two strands are perfectly complementary; for example, if one strand were to have an order of ATGGGCCATA, its complement would be TACCCGGTAT. During replication, the two strands separate, and the base sequence of each strand dictates the construction of a new, complementary strand. In this way, the sequence of the double strands is preserved in the two new identical double strands produced.

A single strand has an orientation that reflects the direction of the phosphodiester bond, which is usually thought of as going from the 5′ to the 3′ end; therefore genes are transcribed in this direction. Because of the double helix structure, the actual transcription occurs from only one strand, called thetemplate strand. The antiparallel strand is referred to as thecoding strand because its base sequence corresponds with the sequence of the message; however, uracil is substituted for thymidine in the message.

The sequence of bases determines the sequence of amino acids in the protein that results from the process of translation. The nucleotides within the coding region are arranged in groups of three, calledcodons, which determine the precise amino acid sequence. Because there are four bases, 64 possible combinations of nucleotides exist, but there are only 20 amino acids. Thus the code is said to bedegenerate, because most amino acids are specified by more than one codon. For example, the code for valine can be GTT, GTC, GTA, or GTG. The third nucleotide can vary for most amino acids and is often called thewobble nucleotide. A specific codon, ATG, codes for methionine and also indicates the beginning of a coding sequence. The three stop codons are TAA, TAG, and TGA.

Abstract

Genetic code refers to the assignment of the codons to the amino acids, thus being the cornerstone template underling the translation process. Genetic code is largely invariant throughout all extant organisms; hence, it is often referred to as the “universal” or “canonical” genetic code. However, a number of extant deviations exist, in both nuclear and organelle (notably, mitochondrial) genomes. These are known as “deviant” or “non-canonical” codes. The emergence of the non-canonical codes posits a number of intriguing questions in regard to the origins and evolution of the universal genetic code and, importantly, has practical implications as certain human mitochondrial diseases have been shown to be linked to the mitochondrial code deviations and translational errors. On a fundamental level, universality (and presumed optimality) of the genetic code is a principal notion underlying its origins, evolution and functionality.

Translation and the Genetic Code

In the cytoplasm, mRNA is translated into protein by the action of a variety of short RNA adaptor molecules, the tRNAs, each specific for a particular amino acid. These remarkable molecules, each only 70 to 100 nucleotides long, have the job of bringing the correct amino acids into position along the mRNA template, to be added to the growing polypeptide chain. Protein synthesis occurs on ribosomes, macromolecular complexes made up of rRNA (encoded by the 18S and 28S rRNA genes), and several dozen ribosomal proteins (seeFig. 3-5).

The key to translation is a code that relates specific amino acids to combinations of three adjacent bases along the mRNA. Each set of three bases constitutes acodon, specific for a particular amino acid (Table 3-1). In theory, almost infinite variations are possible in the arrangement of the bases along a polynucleotide chain. At any one position, there are four possibilities (A, T, C, or G); thus, for three bases, there are 43, or 64, possible triplet combinations. These 64 codons constitute thegenetic code.

Because there are only 20 amino acids and 64 possible codons, most amino acids are specified by more than one codon; hence the code is said to bedegenerate. For instance, the base in the third position of the triplet can often be either purine (A or G) or either pyrimidine (T or C) or, in some cases, any one of the four bases, without altering the coded message (seeTable 3-1). Leucine and arginine are each specified by six codons. Only methionine and tryptophan are each specified by a single, unique codon. Three of the codons are calledstop (ornonsense)codons because they designate termination of translation of the mRNA at that point.

Translation of a processed mRNA is always initiated at a codon specifying methionine. Methionine is therefore the first encoded (amino-terminal) amino acid of each polypeptide chain, although it is usually removed before protein synthesis is completed. The codon for methionine (theinitiator codon, AUG) establishes thereading frame of the mRNA; each subsequent codon is read in turn to predict the amino acid sequence of the protein.

The molecular links between codons and amino acids are the specific tRNA molecules. A particular site on each tRNA forms a three-baseanticodon that is complementary to a specific codon on the mRNA. Bonding between the codon and anticodon brings the appropriate amino acid into the next position on the ribosome for attachment, by formation of a peptide bond, to the carboxyl end of the growing polypeptide chain. The ribosome then slides along the mRNA exactly three bases, bringing the next codon into line for recognition by another tRNA with the next amino acid. Thus proteins are synthesized from the amino terminus to the carboxyl terminus, which corresponds to translation of the mRNA in a 5′ to 3′ direction.

1 Overview of orthogonal translation systems in genetic code expansion

GCE encompasses numerous techniques that allow co-translational installation of ncAAs into proteins within living organisms. Two general strategies have been developed for achieving GCE. In one strategy, ncAAs that are isostructural analogs of canonical amino acids (cAAs) are incorporated into proteins by endogenous aaRSs, which are unable to distinguish between the analog and their natural substrate. Using this methodology, known as residue-specific GCE or sense codon reassignment, all instances of the AA within a protein may be replaced by the non-canonical analog. In the second strategy, known as site-specific GCE or stop codon suppression, exogenous aaRS and tRNA pairs are expressed within a host organism and facilitate the incorporation of ncAAs in response to reassigned codons. Typically, site-specific GCE utilizes nonsense suppressor tRNAs that introduce the ncAA in response to reassigned stop codons. Therefore, unlike residue-specific GCE, with site-specific GCE the position of the ncAA within a protein can be precisely defined by introducing a nonsense mutation into the protein coding gene.

To be useful for site-specific GCE an aaRS•tRNA pair must fulfill the following criteria: (1) the aaRS must be able to be expressed in its active form within the host organism, (2) the tRNA must be correctly processed within the host organism, (3) the tRNA must be compatible with the translational machinery (ribosome, elongation factors, etc.) of the host, (4) the aaRS•tRNA pair must not cross-react with endogenous aaRSs and tRNAs, i.e., it must be orthogonal, and (5) the aaRS must selectively recognize an ncAA substrate over cAAs. In this chapter we describe several aaRS•tRNA pairs that meet these criteria and have been used to site-specifically install ncAAs into proteins in various host organisms.

DNA and the genetic code

Hereditary information is contained in the sequence of the building blocks of double-stranded DNA (Fig. 2.4). Each strand of DNA is made up of a deoxyribose–phosphate backbone and a series of purine (adenine (A) and guanine (G)) and pyrimidine (thymine (T) and cytosine (C)) bases. Because of the way in which the sugar–phosphate backbone is chemically coupled, each strand has a polarity, with a phosphate at one end (the 5′ end) and a hydroxyl at the other (the 3′ end). The two strands of DNA are held together by hydrogen bonds between the bases. T can pair only with A, and G can pair only with C; therefore, each strand is the antiparallel complement of the other (seeFig. 2.4). This is key to DNA replication because each strand can be used as a template to synthesize the other.

The two strands twist to form a double helix with a major and a minor groove, and the large stretches of helical DNA are coiled around histone proteins to form nucleosomes (seeFig. 2.4). They can be condensed further into the chromosomes that can be visualized by light microscopy at metaphase (seeFigs 2.4 and2.11).

Ribonucleotide Bases Are Used as Letters in the Genetic Code

The genetic code is written in linear form, using the ribonucleotides that compose mRNA molecules as letters. The ribonucleotide sequence is derived from the complementary nucleotide bases in the DNA template strand. Therefore, the nucleotide sequence is exactly the same as the DNA coding strand. Each genetic code consists of three ribonucleotide letters, thus referred to as a triplet code. As such, a genetic code is a triplet code in which a sequence of three bases is needed to specify one amino acid. The genetic code translates the RNA sequences into the amino acid sequence (Fig. 4.17). Each group of three ribonucleotides, called a codon, specifies one amino acid. These codes are unambiguous, as each triplet specifies only a single amino acid. Thus, one would imagine that a codon would be at least three bases long. With three bases, there are 43 = 64 codons, which is more than enough to encode the 20 amino acids. Therefore, the genetic code is degenerate, which means more than one triplet can encode the same amino acid. Each amino acid can have more than one codon, but no codon can encode more than one amino acid. Furthermore, the genetic code is universal, as the code can be used by all viruses, prokaryotes, archaea, and eukaryotes.

1.15 Degenerate primers

A PCR primer sequence is called ‘degenerate’ if some of its positions have several possible bases. The ‘degeneracy’ of the primer is the number of unique sequence combinations it contains. Hence, these primers are supplied as mixtures of similar, but not identical, oligonucleotides. They have various applications such as amplification or cloning of related genes from the same or different organisms, cloning of genes based on protein sequence, or genome walking with techniques such as TAIL-PCR (thermal asymmetric interlaced PCR, Liu and Whittier, 1995).

Sequence comparison of multiple members of protein families has revealed protein motifs and domains that play important roles in protein function. Closely related sequences can be cloned using degenerate primers (a pool of primers containing most, or all, possible nucleotide sequences encoding a conserved amino acid motif) or consensus primers (a single primer containing the most common nucleotide at each codon position within the motif).

The goal is then to design primers that match as many sequences of homologous genes as possible. A naïve solution would be to align the sequences without gaps, count the number of different nucleotides in each position along the alignment, and seek a primer-length window (20–30 bp) where the product of the count is low. Such a solution is insufficient because of gaps, the inappropriate objective function of the alignment, and most notably, the exceedingly high degeneracy: when degeneracy is too high, unrelated sequences may be amplified as well, losing specificity (Linhart and Shamir, 2002; 2005). The degeneracy has to be high to maximize the coverage, but has to be bound to decrease the probability of amplifying unrelated sequences. This problem and the approaches to solve it are addressed in Najafabadi et al. (2008).

Below, some software approaches are suggested for the design of degenerate primers:

PAMPS (Pairwise Alignment for Multiple Primer Selection). In contrast to previous algorithms, this one does not restrict the output to the exact primer length that was given, which allows selecting an appropriate primer in terms of annealing temperature. PAMPS can be used to design degenerate primers for amplifying genes with uncertain sequences, such as new members of gene families or libraries of antibody variable fragments. PAMPS has been shown to outperform previous algorithms (HYDEN and MIPS) in this task. This software is freely available.

Other tactics have been developed based on COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs). A CODEHOP is a hybrid primer consisting of a 15–20 nt consensus 5′-clamp and a 9–12 nt degenerate 3′-core region. The core gives a broader specificity for distantly related target gene templates, while the clamp allows for a robust amplification during the later cycles of the PCR. CODEHOP works well for small sets of proteins, taking into account the codon usage of the target genome and the desired annealing temperature. However, it is inappropriate for constructing primers with very high degeneracy on large sets of long genomic sequences, and software like PAMPS should be used for that task. An interactive version of the software, iCODEHOP, is freely available.

MAD-DPD (Minimum Accumulative Degeneracy Degenerate Primer Design). MAD-DPD can be considered as a first approach in primer design when a high degeneracy is desired and the number of degenerate primers to be constructed is known or has to be kept low. A drawback of this software is that it does not consider parameters like melting temperature, self-annealing, or secondary structures. This software is freely available.

MIPS (Multiple, Iterative Primer Selector). The software MIPS outperforms HYDEN on the task of designing multiplex PCR experiments for SNP genotyping. This software is freely available.

HYDEN (HighlY DEgeNerate primers) is a software useful for constructing primers and primer pairs with high degeneracy and yet high specificity. This software is freely available.

Use of degenerate primers can greatly reduce the specificity of the PCR amplification, a problem that can be partly solved by using touchdown PCR.

Genetic code (IUPAC)

4 Conclusion and perspectives

Genetic code reprogramming technology has proven to be powerful not only to express nonstandard peptides, but also to discover de novo potent inhibitors and activators of proteins of interest upon integration with mRNA display, referred to as the RaPID system. In this chapter, we have described two of the most recent technologies developed in our research group. One of the technologies, relying on the affinity tuning of MeAA-tRNAs to EF-Tu, has significantly improved their incorporation efficiency into the nascent peptide chain, leading to expression with higher quantity and fidelity. This technology enables us to express a library of highly N-methylated backbone-modified macrocyclic peptides, leading us to discover more peptidase-resistant and cell membrane permeable molecules. Post-translation installation of azole groups described as the following technology also adds a unique functionality to ordinary peptide sequences, which potentially gives the same impact on their drug-like properties. These technologies can be combined, and thus further enhance peptide diversity and functionality. The advancement of this field is quite rapid lately, so that we are certain that peptide modalities will become a reliable pipeline of therapeutic development.

Genetic Code

The genetic code is the universal dictionary by which genetic information is translated into the functional machinery of living organisms, the proteins. The words or ‘codons’ of the genetic message are three nucleotides long. Since there are four different nucleotides used in messenger RNA (mRNA), this results in a dictionary of 64 words. There are 20 amino acids that are normally used in proteins and which are translated. In addition the translation needs a definition of ‘start’ and ‘stop.’ The start codon defines the start of translation as well as the reading frame (the sequence of nucleotide triplets) that is to be translated. The start or initiator codon is identical to the methionine codon. Special mechanisms are used to identify the correct initiation site; in addition there are three stop codons. Thus 61 codons are available for 20 amino acids, and hence the genetic code is degenerate. In the case of leucine, serine, and arginine, there are as many as six codons, whereas methionine and tryptophan have only one codon.

The universal genetic code deviates slightly in mitochondria, where a few codons are translated in alternative ways. The most prevalent are methionine and tryptophan, which have two codons instead of the usual one. Different organisms use the degenerate genetic code differently. The usage of the codons is coupled to the availability to tRNAs that can translate them. Thus the codon usage can differ to the extent that a gene that is transferred from one organism to another cannot be translated unless the new organism is supplemented with extra tRNAs.

4 Synthetic Chemistry-Guided Unnatural Amino Acid Design

Genetic code expansion enables the usage of unnatural chemical groups, which are widely used in organic chemistry but is rare in organisms. At least three advantages can be achieved by doing that. First, the protein scaffold provides a secondary coordination sphere for the organic catalyst, which may enhance their performance, including turnover numbers and enantioselectivity (Durrenberger & Ward, 2014). Second, the structure containing unnatural organic amino acid is genetically encoded. As a consequence, its self-assembly can be easily amplified or improved by directed evolution. Finally, adding unnatural organic molecules enables researchers to solve biological chemistry problems by organic chemical methods, which may be helpful in green chemistry and synthetic biology.

Some unnatural amino acids were inspired by organic chemistry studies. Thus, some unnatural organic molecules and powerful and highly developed synthetic chemistry methods can be introduced to molecular biology (Mann, 1989). In order to be properly incorporated, those organic molecules are required to be converted into an unnatural amino acid first. Based on their structure features, they can be converted into either a “tyrosine” or a “lysine”. For instance, if the molecule contained an aromatic ring, it would be suitable for mimicking tyrosine. The tyrosine type unnatural amino acid often contains an aromatic ring that bears the unnatural chemical groups, and a covalently linked aliphatic amino acid part (usually alanine) as the amino acid back bone. On the other hand, a lysine host is more suitable for flexible aliphatic chemical groups. The lysine mimic is usually composed of the unnatural aliphatic chemical groups and a lysine molecule. Usually they are covalently connected by an amide bond or carbamate.