A minimal cell containing biological macromolecules and pathways proposed to be necessary and sufficient for replication from small molecule nutrients. The macromolecules are all nucleic acid and protein polymers and are encapsulated within a bilayer lipid vesicle. The small molecules (brown) diffuse across the bilayer. The macromolecules are ordered according to the pathways in which they are synthesized and act. They are colored by biochemical subsystem as follows: blue=DNA synthesis, red=RNA synthesis and cleavage, green=RNA modification, purple=ribosome assembly, orange=post-translational modification and black=protein synthesis. MFT=methionyl-tRNA^fMet_i formyltransferase. The system could be bootstrapped with DNA, RNA polymerase, ribosome, translation factors, tRNAs, MTF, synthetases, chaperones and small molecules.

A generalizable, physiologically compatible, theoretical scheme for accurate DNA replication and RNA synthesis in vitro. Polymerase movements are illustrated by colored arrowheads. DNA synthesis: a nicked double-stranded DNA circle (middle) undergoes rolling-circle DNA synthesis by coliphage φ29 DNA polymerase (Dahl et al, 2004) to give an oligomeric single-stranded DNA (bottom, blue). RNA primers (red) then hybridize at two sites to prime lagging strand DNA synthesis (bottom, green). When two Lox sites (bottom, L) are completed, recombination occurs between them catalyzed by coliphage P1 Cre recombinase (black cross) to form a duplicate of the original circular template. RNA synthesis: the circular genetic operon (middle) contains a promoter for T7 RNA polymerase (P), a ribosomal RNA (rRNA) gene, two transfer RNA (tRNA) sequences, a self-cleaving hammerhead sequence (H) and a T7 terminator (T). RNA synthesis from P generates a precursor RNA (top, red) containing three cleavage sites (thin black arrows). The second tRNA sequence merely serves as a recognition site for RNase P cleavage. Cleavages yield the mature rRNA and tRNA₁. Any cleavage product containing a 3′ hydroxyl group or primary RNA transcript can serve as a primer for DNA synthesis (bottom, red).

All nucleoside modifications of all 33 synthetic tRNAs that may be sufficient for accurate translation. Outside (shaded): mRNA codons of the genetic code are illustrated in the standard format, except that the 3′ U and C are switched to simplify depiction of decoding. Inside: tRNA nucleotides 34–37 (from 5′ to 3′) and their cognate amino acids. Nucleotides 34–36 are the anticodons, and the 37th nucleotides are represented by black superscripts. Codon and anticodon positions that base pair with each other are colored similarly. Stop codon specificities of release factor (RF) proteins are included. The portions of the tRNA sequences not shown in the figure are unmodified. Expected modifications of in vitro transcripts by the enzymes in Table I, and expected amino-acid and codon specificities are given. ^*=unspecified modification, _=unknown modification status, ms²i⁶A=2-methylthio-N⁶-isopentenyladenosine, m¹G=1-methylguanosine, t6A=N⁶-threonylcarbamoyladenosine, cmnm⁵U=5-carboxymethylaminomethyluridine, V=cmo⁵U=uridine 5-oxyacetic acid, I=inosine, cmnm⁵s²U=5-carboxymethylaminomethyl-2-thiouridine, k2C=lysidine, S=mnm⁵s²U=5-methylaminomethyl-2-thiouridine, mnm⁵U=5-methylaminomethyluridine.