Streptomycin-Suppressible Lethal Mutations in Escherichia coli

Forty-one mutants have been isolated which require streptomycin for growth on complete medium. These streptomycin-suppressible lethal mutations are located randomly around the Escherichia coli genetic map; during growth in liquid culture, they exhibit a variety of responses to the removal of streptomycin as judged by turbidity, cell morphology, and macromolecular synthesis. In particular, some mutants are primarily affected in protein or ribonucleic acid (RNA) synthesis (or both), one in deoxyribonucleic acid synthesis, and two in lipid synthesis. Ten mutants affected in protein synthesis were examined for the activities of all twenty aminoacyl-transfer RNA synthetases, and three were found to have altered glutamyl-transfer RNA synthetase activities. The advantages of this method for isolating a wide variety of conditional lethal mutants are discussed.     

Hypothesis: Emergence of Translation as a Result of RNA Helicase Evolution

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.     

Prebiotic chemistry and the origin of the RNA world

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, we review studies of prebiotic membrane formation.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

Prebiotic Chemistry and the Origin of the RNA World

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have “invented” RNA. Finally, we review studies of prebiotic membrane formation.     

Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA. We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein.     

Photosynthesis and the Origin of Life

The origin and evolution of photosynthesis is considered to be the key to the origin of life. This eliminates the need for a soup as the synthesis of the bioorganics are to come from the fixation of carbon dioxide and nitrogen. No soup then no RNA world or Protein world. Cyanobacteria have been formed by the horizontal transfer of green sulfur bacterial photoreaction center genes by means of a plasmid into a purple photosynthetic bacterium. The fixation of carbon dioxide is considered to have evolved from a reductive dicarboxylic acid cycle (Chloroflexus) which was then followed by a reductive tricarboxylic acid cycle (Chlorobium) and finally by the reductive pentose phosphate cycle (Calvin cycle). The origin of life is considered to have occurred in a hot spring on the outgassing early earth. The first organisms were self-replicating iron-rich clays which fixed carbon dioxide into oxalic and other dicarboxylic acids. This system of replicating clays and their metabolic phenotype then evolved into the sulfide rich region of the hotspring acquiring the ability to fix nitrogen. Finally phosphate was incorporated into the evolving system which allowed the synthesis of nucleotides and phospholipids. If biosynthesis recapitulates biopoesis, then the synthesis of amino acids preceded the synthesis of the purine and pyrimidine bases. Furthermore the polymerization of the amino acid thioesters into polypeptides preceded the directed polymerization of amino acid esters by polynucleotides. Thus the origin and evolution of the genetic code is a late development and records the takeover of the clay by RNA.     

The Early Phases of Genetic Code Origin: Conjectures on the Evolution of Coded Catalysis

A review of the most significant contributions on the early phases of genetic code origin is presented. After stressing the importance of the key intermediary role played in protein synthesis, by peptidyl-tRNA, which is attributed with a primary function in ancestral catalysis, the general lines leading to the codification of the first amino acids in the genetic code are discussed. This is achieved by means of a model of protoribosome evolution which sees protoribosome as the central organiser of ancestral biosynthesis and the mediator of the encounter between compounds (metabolite-pre-tRNAs) and catalysts (peptidyl-pre-tRNAs). The encounter between peptidyl-pre-tRNA catalysts in protoribosome is favoured by metabolic pre-mRNAs and later resulted (given the high temperature at which this evolution is supposed to have taken place) in the evolution of mRNAs with codons of the type GNS. These mRNAs codified only for those amino acids that the coevolution theory of genetic code origin sees as the precursors of all other amino acids. Some aspects of the model here discussed might be rendered real by the transfer-messenger RNA molecule (tmRNA) which is here considered a molecular fossil of ancestral protein synthesis.     

Nontemplate polymerization of free nucleotides into genetic elements by thermophilic DNA polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life.     

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.     

Glyoxylate as a Backbone Linkage for a Prebiotic Ancestor of RNA

The origin of the first RNA polymers is central to most current theories for the origin of life. Difficulties associated with the prebiotic formation of RNA have lead to the general consensus that a simpler polymer preceded RNA. However, polymers proposed as possible ancestors to RNA are not much easier to synthesize than RNA itself. One particular problem with the prebiotic synthesis of RNA is the formation of phosphoester bonds in the absence of chemical activation. Here we demonstrate that glyoxylate (the ionized form of glyoxylic acid), a plausible prebiotic molecule, represents a possible ancestor of the phosphate group in modern RNA. Although in low yields (∼ 1%), acetals are formed from glyoxylate and nucleosides under neutral conditions, provided that metal ions are present (e.g., Mg²⁺), and provided that water is removed by evaporation at moderate temperatures (e.g., 65 ^∘C), i.e. under “drying conditions”. Such acetals are termed ga-dinucleotides and possess a linkage that is analogous to the backbone in RNA in both structure and electrostatic charge. Additionally, an energy-minimized model of a gaRNA duplex predicts a helical structure similar to that of A-form RNA. We propose that glyoxylate-acetal linkages would have had certain advantages over phosphate linkages for early self-replicating polymers, but that the distinct functional properties of phosphoester and phosphodiester bonds would have eventually lead to the replacement of glyoxylate by phosphate.     

Regulation of RNA function by aminoacylation and editing?

The chemical modification of nucleic acids is a ubiquitous phenomenon. Aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a reaction essentially devoted to protein synthesis but it is used also as an emergency mechanism to recycle stalled ribosomes, and it is required for genome replication in some RNA viruses. In several aminoacyl-tRNA synthetases a correction mechanism known as editing is present to prevent aminoacylation errors. Genome data reveal a growing number of open reading frames encoding ARS-like proteins. This strongly suggests the existence of a widespread and nonconventional machinery for aminoacylation and editing. Here we review the different biological functions of aminoacylation and editing; also we propose an evolutionary scenario for the origin of these two reactions, and hypothesize an extant role for RNA charging and editing outside the genetic code.     

Origins of tmRNA: the missing link in the birth of protein synthesis?

The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.