Before the sequencing of DNA became routine, animals and plants were classified reasonably well, fungi and other primitive eukaryotes were classified poorly, and bacterial classification was a lost cause due to lack of observable characters. Using gene sequences for classification was developed for bacteria and has since spread to other types of organism. Nowadays ancestries may be traced by comparing the sequences of DNA, RNA or proteins that are more representative of fundamental genetic relationships than are many superficial characteristics. Furthermore, in situations where division into species, genera, families, etc., is arbitrary, sequence data can provide
Consider an essential molecule that evolves slowly, such as histones or ribosomal RNA. It is possible that certain combinations of two mutations might yield a functional molecule, but that either alone would be lethal. For example, a mutation from G to C that destroyed a critical GC base pair in a stem loop structure might be fatal in 16S rRNA. However, replacing the GC with a CG base pair might well allow function (Fig. 20.19). During normal evolution, this replacement is highly unlikely since either single mutation is lethal and the likelihood of simultaneous mutations in just these two bases is very low.
Consequently, a CG base pair in this particular position will probably be very rare among the 16S rRNA sequences of existing life forms. To fully analyze the relationship of structure and function in a molecule such as rRNA, several artificial mutations must be introduced simultaneously. This may be done by a procedure known as "instant evolution" that was developed in the laboratory of Dr. Philip R. Cunningham at Wayne State University. In this approach, 16S rRNA is mutated and those mutations that prevent protein synthesis are isolated. Next, suppressor mutations that restore protein synthesis are selected. Alternatively, multiple random mutations may be simultaneously introduced into a short region of the rRNA that is suspected to play an important role in protein synthesis. In either case, most of these mutations in rRNA would be lethal under normal circumstances; to avoid killing the bacteria they must be manipulated so that the mutant form of the 16S rRNA does not interfere with normal cellular protein synthesis.
The following technology was developed to prevent the mutated form of rRNA from affecting the normal bacterial functions (Fig. 20.20):
Mutations in ribosomal RNA are lethal since they alter the stem-loop structure of the molecule. In this example, mutating the guanine to cytosine prevents a critical base pair from completely the stem portion. If a second mutation were to change the cytosine to a guanine, the base pair would reform and the ribosomal RNA would be functional again. Although changing both positions simultaneously is extremely unlikely, this would not be detrimental to the organism and the mutation would be passed onto successive generations.
Normal cellular pioLfins
FIGURE 20.20 Instant Evolution of Ribosomal RNA
Normal cellular pioLfins
The plasmid pRNA122 carries the sequence for altered 16S rRNA and the reporter gene (in this example, CAT). Separation of chromosomal and plasmid-directed protein synthesis occurs primarily due to the changes in the Shine-Dalgarno (SD) sequence. The 16S rRNA encoded by the plasmid cannot recognize the SD sequence of normal cellular mRNA, but does recognize the SD sequence upstream of the reporter gene (cat). If a mutation in the 16S rRNA prevents it from functioning, the reporter gene is not translated into CAT protein, and therefore the bacteria are not chloramphenicol resistant. Translation of normal cellular proteins occurs without interruption because the chromosomal copy of the 16S rRNA (small pink oval) is used. The chromosomal copy of the 16S rRNA does not recognize the SD sequence of the cat mRNA, and so does not allow synthesis of CAT protein.
a. A copy of the 16S rRNA gene was put on a plasmid and mutated. Since the genomic copy of the 16S rRNA is still functional, most of the cell's ribosomes will be normal. Only a fraction of the ribosomes will have the mutant 16S rRNA.
b. The anti-Shine-Dalgarno sequence of the plasmid 16S rRNA is altered so that it no longer recognizes normal cellular mRNA, thus lethal mutations in this 16S rRNA copy will not interfere with normal protein synthesis.
c. A reporter gene is designed with an altered Shine-Dalgarno sequence that matches the plasmid or mutated form of the 16S rRNA. Thus only the translation of the mRNA from the reporter gene responds to the mutations in the plasmid-borne copy of the 16S rRNA. Two different reporter genes were used, chloramphenicol acetyl transferase (CAT), which gives chloramphenicol resistance to the bacteria, and green fluorescent protein (GFP), which causes the bacteria to turn green under fluorescent light. The mutant 16S rRNA is therefore functionally isolated from the rest of the cell and can be analyzed by monitoring the expression of the two proteins CAT and GFP. Lethal mutations in 16S rRNA merely prevent expression of CAT and GFP without affecting the normal protein synthesis of the bacteria.
In these experiments nearly 60,000 different SD-anti-SD combinations were tried but only 13 were found to be functional without killing the cell. Researchers in the Cunningham laboratory showed that almost any change in the nucleotide sequence of the anti-SD was lethal to bacteria-probably because it disrupted the balance of protein synthesis. Since its development, instant evolution has been used by several researchers throughout the world to study the role of ribosomal RNA in protein synthesis. This technology may also allow the development of new antibiotics targeted against critical regions of the ribosome and that are not susceptible to the development of drug resistance.
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