Genetic Code Mutation

Many amino acid analogues are readily incorporated into prokaryotic and eukaryotic proteins, but they invariably fail to support indefinite cell growth. To test Tenet 4 of CET, experiments were conducted in 1983 to mutate the amino acid code of B. subtilis QB928 (a Trp-auxotroph that cannot revert out of its Trp-dependence) in order to replace the use of Trp for long term cell growth by its analogue 4-fluoroTrp (4FT). In two mutational steps, QB928 gave rise to strain LC33 (LC = large colonies on 4FT plates), which can grow indefinitely on 4FT forming colonies on agar (Fig. 14.4). Although two mutational steps might be accompanied by more than two mutations, the number of mutations required to arrive at 4FT utilization were still surprisingly few. When LC33 was further mutated, it gave rise to HR15 in another two mutational steps (HR = high 4FT/Trp growth ratio), which grows well in 4FT but not on Trp. Thus Trp is replaceable by 4FT in LC33. In HR15 and its derivative strains such as faster growing HR23 and Met-independent MR3, Trp has in fact been displaced from the amino acid code: it can no longer support long term cell growth. Growing on 4FT, these strains are inhibited by Trp now acting as an inhibitory analogue. However, the cells can back mutate to enable Trp to regain its lost growth-support capacity (Fig. 14.5). LC33, which grows on Trp and 4FT, has also yielded through mutations the LC88 strain, which grows on Trp, 4FT, 5-fluoroTrp (5FT) and 6-fluoroTrp (6FT) (Fig. 14.4), even though 5FT is a potent inhibitor of bacterial growth.59-61

The fall ofTrp from its status as a canonical amino acid in HR15 indicates that genetic code evolution was by no means limited to the addition of novel amino acids to the code. Amino acids also could be tried out, found wanting and rejected from the code. This type of turnover would explain the otherwise inexplicable absence

Figure 14.5. Inhibition by Trp placed in center well against B. subtilis MR3 growing on 4FT. Revertant colonies that have regained the ability to grow on Trp are visible within the cleared Trp-inhibition zone.61

Figure 14.5. Inhibition by Trp placed in center well against B. subtilis MR3 growing on 4FT. Revertant colonies that have regained the ability to grow on Trp are visible within the cleared Trp-inhibition zone.61

Box 14.2. Seatings at Wedding Reception

The distribution of codons to different amino acids may be compared to the seating ofguests at a wedding reception. There are 20 guests,

A, B, S,T, and there are twenty seats. There will be a total of p!(q!)p different seating arrangements, where p is the number of seating sections, and q is the number of heads per section. In the first seating approach, all 20 guests are treated as a single group, drawing lots to determine seat assignment, so that p = 1 and q = 20. This yields a total of 2.4 x 1018 different seating arrangements for the guests.

Since there are five affinity groups of guests, four per group — (1) A-D are bride's relatives, (2) E-H are groom's relatives, (3) I-L are bride's coworkers, (4) M-P are groom's coworkers, and (5) Q-T are neighbors, a second seating approach is to divide the seats into five sections, and randomly draw lots to allocate these sections to groups 1-5. The four seats within each section will be randomly distributed to the four individuals within the same group. In this case, p = 5 and q = 4. This yields a total of only 9.6 x 108 seating arrangements. Thus the constraint imposed in the second approach disallowing all mixed-group seating within the same section reduces the number of possible seating arrangements by a factor of 4 x 10-10.

Likewise, when the 64 codons in the genetic code are distributed to the 20 amino acids and termination signal without any affinity grouping, the number of possible codes differing in codon allocations is approximately 2 x 1019. If the 20 amino acids are divided into affinity groups based on biosynthetic relationships, the number of possible codes becomes 2 x 108. This reduction of allowable alternate codes by a factor of 10-11 greatly facilitates the selection of a unique code out of all the possible codes.

of a-aminobutyric acid from the code. This amino acid is readily produced by atmospheric abiotic synthesis. In the electric spark synthesis in Table 9.1, the amount of a-aminobutyric acid produced was less than Gly and Ala, but more than Glu and Asp. Its absence from the genetic code to-day is likely due to initial try-out by the life forms followed by rejection. Being a 4-carbon hydrophobic amino acid, it might have lost out to amino acids like Val, Leu and Ile which offer a more bulky alkyl sidechain and stronger hydrophobicity.

The relatively small number of mutational steps employed to achieve replacement or displacement of Trp from the code by its fluoro-analogues suggests that there are a small number of Trp residues among the 12,625 Trp residues of the B. subtilis proteome where the protein structure around the residue is so restrictive that Trp replacement by 4FT, 5FT or 6FT leads to defective function and therefore growth inhibition. When the structural constraint is relaxed around these critical residues, replacement of Trp by 4FT, 5FT or 6FT is tolerated, as in LC88. However, if the structural constraint is altered in such a manner that it restricts protein function with Trp but not with 4FT, it would cause Trp to lose its growth-support capacity and become an inhibitory analogue.

The introduction of4FT into the encoded amino acids ofB. subtilis represents the first known instance of a genetic code mutation altering the ensemble of canonical amino acids. It provides decisive support for Tenet 4 ofthe coevolution theory and establishes that, in spite ofthe freezing ofthe genetic code with respect to its canonical amino acids since the earliest branchings of living species, the code remains intrinsically mutable. The thawing of this once frozen code has ushered in a new era of genetic code mutations. There are two mutational approaches. The top-down or genome wide approach adopted for the encoding of 4FT in B. subtilis has been extended to E. coli and coliphage.63-65 In the bottom-up approach, special orthogonal aaRS-tRNA pairs are employed to place an unnatural amino acid into selected positions in protein sequences.66,67 These orthogonal pairs, based on the lack of reactivity between cross domain aaRS-tRNA pairs,68 interact minimally with other aaRS and tRNAs inside the cell.

Because the amino acid alphabet has been a fundamental, unchanging attribute of all living organisms, the new organisms bearing novel encoded amino acids may be regarded as new forms of life69 that are transforming the biology of the past into a sequel where straitjacketing by the traditional twenty encoded amino acids is relaxed to allow increased dimensions of freedom in evolution based on wider variations in the amino acids.70 Bacillus subtilis strain HR15, which grows on 4FT but not Trp as a canonical encoded amino acid, thus represents the first example of synthetic bilogy.

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