Lysine codons

Another possible application of suppressor genes is in vivo suppression of undesirable termination codons. An example comes from a p° thalassemia caused by mutation of lysine codon CAG to UAG. By changing the anticodon of a human tRNALys gene to... 

This is a lysyl tRNA synthetase enzyme in the first instance that should have the expected capacity to couple the naturally available amino acid L-lysine to appropriate cognate tRNAs bearing anti-codon sequences complementary to lysine codons (see Section 1.6.1). However in the presence of zinc ions, Zn +, the function of this enzyme becomes altered to catalyse the biosynthesis of diadenosine-5, (Ap4A) followed by... 

The expression plasmid pDS12/RBSII was adapted for expression by a synthetic oligonucleotide (dotted line). The cDNA (14)(a kind gift of R.G. Herrmann, Munich) was cloned into the Hindlll cleaving site within the lysine codon. The amino acid sequence shows the resultant signal sequence. The bracket indicates the Shine-Dalgarno sequence. 

The many (possibly more than 30) types of collagens found in human connective tissues have substantially the same chemical structure consisting mainly of glycine with smaller amounts of proline and some lysine and alanine. In addition, there are two unusual amino acids, hydroxyproline and hydroxylysine, neither of which has a corresponding base-triplet or codon within the genetic code. There is therefore, extensive post-translational modification of the protein by hydroxylation and also by glycosylation reactions. 

Table 12.1. The genetic code presented in this table is very nearly universal. There are isolated exceptions in the genome of mitochondria, which is described later in this chapter. Beyond that, the genetic code has been expanded to include codons for two unusual amino acids that occur in a modest number of proteins. These amino acids are selenomethionine, in which an atom of selenium replaces the sulfur atom of methionine, and pyrrolysine, a cychzed form of lysine. For details, see A. Ambrogelly, S. Palioura, and D. Soil, Nat Chem Biol 3 29-35 (2007). 

In the next step, translation, the sequence of nucleotides in the newly synthesized mRNA strand is used to determine the sequence of amino acids in the protein to be synthesized. This is done by way of a genetic code, which was fully deciphered by 1966 and is shown in Figure 13.34. According to the genetic code, it takes three mRNA nucleotides—each three-nucleotide unit is called a codon—to code for a single amino acid. The mRNA nucleotide sequence AGU, for example, codes for the amino acid serine, and AAG codes for lysine. (Note from Figure 13.34 that more than one codon can call for the same amino acid.) A few codons, such as AUG and UGA, are the signals for protein synthesis to either start or stop. 

It is quite certain that the code involves a particular sequence of three nucleotides for each amino acid. Thus the sequence A-A-A codes for lysine, and U-C-G codes for serine. The sequences or codons for all twenty amino acids are known. 

Figure 25-28 Peptide-bond formation in protein biosynthesis showing how the amino-acid sequence is determined by complementary basepairing between messenger RNA and transfer RNA, The peptide chain is bound to tRNA, which is associated with mRNA through three bases in mRNA (codon) and three bases in tRNA (anticodon). In the diagram, the next codon A-A-G codes for lysine. Hence, Lys-tRNA associates with mRNA by codon-anticodon base-pairing and, under enzyme control, couples to the end of the peptide chain.

The codons of the mRNA on the ribosomes are read from the 5 to the 3 end. Thus the synthetic polynucleotide (5 )A-A-A-(A-A-A) -A-A-C(3 ) contains the code for lysine (A-A-A) and asparagine (A-A-C) the actual polypeptide obtained using this mRNA in a cell-free system was Lys-(Lys),-Asn, and not Asn-(Lys) -Lys. 

Selective formation of microparticles from polynucleotides and lysine-rich proteinoids rich in individual radioactive amino acid has been studied and the focus of attention is on those homoanticodonic amino acids having one homogeneous codon (glycine, CCC lysine, UUU proline, GGG and phenylalanine, AAA)58). Precipitation of individual amino acid rich proteinoids with each of homopolyribonucleotides, with and without Mg2+, was tested58>. The results show that three (Lys-rich, Gly-rich,... 

Microparticles composed of each of four homopolyribonucleotides and the same lysine-rich proteinoid is found to influence the incorporation of individual amino-acyl adenylate 59). The incorporation favors the amino acids whose codons are related to the nucleotide in the particles (Fig. 6), when conditions are appropriately chosen. Other conditions yield other preferences. These results support a stereochemical basis for the genetic code. 

Few polymorphisms have been detected in the human AHR gene. Thus far only one human AHR polymorphism has been shown to exert a substantial effect on receptor function human AHR that encodes serine at codon 517, when in combination with isoleucine at 570 and lysine at 554 (a presumed haplotype found only in individuals of African descent), fails to sustain CYP1A1 induction in vitro even though this variant is competent to bind the inducer, TCDD, and to bind AH response elements derived from the CYP1A1 gene (80). 

Both ornithine, which is a homologue of lysine, and citrulline are L-amino acids, but neither has a genetic codon, and both are found only as posttranslational modifications of arginine residues in some proteins such as keratin. Citrulline leaves the mitochondria by the same transport system that facilitates ornithine s entry from the cytoplasm. 

Figure 75. Alignment of lysin and 18K proteins from red abalone. Both proteins have five exons (numbered) and four introns (positions shown by arrows). The position of intron three in lysin could not be determined and is shown by a question mark corresponding to the third 18K intron. For both proteins, the known introns are in the exact same positions and have the same phase in the interrupted codon (phase numbered above arrow head). These data indicate the two proteins arose by gene duplication.

When the lysin sequences of the first seven species were obtained, we were impressed at how much divergence had occurred between their primary structures (Figure 9 Table 1). We also discovered that amino acid replacement was mainly nonconservative regarding the class of residue replaced. Next, we made pairwise comparisons of the aligned cDNA sequences and scored the numbers of amino acid altering (nonsynonymous) and silent (synonymous) nucleotide changes in the 21 pairwise comparisons of the seven sequences. The data (Table 2) showed that the vast majority of codon differences between any two lysins are amino acid altering. For example, in the comparison of mature red and pinto abalone lysins of 136 codons, 25 of the codon differences are nonsynonymous and only one is silent (Lee and Vacquier, 1992). 

Table 2. Ratio of Amino Acid Altering to Silent Codon Differences in Pairwise Comparisons of Mature Lysins (136 Codons)...

---