Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage.

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation.

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2). magnesium chelatase mutant ͉ photosynthetic bacterial reaction center ͉ photosystems I and II ͉ protein-cofactor interaction T he purple bacterial reaction center (RC) is a pigmentprotein complex that is capable of converting light energy to chemical energy with quantum yield approaching 1 (1-3). Electron transfer (ET) in this RC has been extensively studied; the structure and spectroscopic features of the complex are well known, the complex is very stable, and a large variety of mutants is available. This RC also serves as a model system for understanding protein-cofactor interactions and the role that protein plays in ET (4).The RC from Rhodobacter (Rb.) sphaeroides comprises 3 protein subunits, H, M, and L. As shown in Fig. 1, the RC complex binds 9 cofactors that form 2 potential ET chains (referred to as A and B) in a C2 symmetric arrangement. The ''special pair'' (P) is a dimer of bacteriochlorophyll (BChl) a molecules and is located on the periplasmic side of the cytoplasmic membrane. Two monomeric BChls (B A and B B , with the subscripts denoting which chain the cofactor belongs to) are present on either side of P. These are followed by 2 bacteriopheophytin (BPhe) molecules (H A and H B ). A nonheme iron and 2 quinones (Q A and Q B ) are near the cytoplasmic side of the RC (5, 6). When P is excited, an electron is transferred through the A branch cofactors, and then to Q B . In the WT RC, the times for ET from P* to H A to Q A to Q B are 3 ps, 200 ps, and 200 s, respectively. The transfer from P* to H A is thought to be via B A .The ET reactions P...

Nonresonant and resonant transient, photochemical hole-burned (HB) spectra are presented for primary electron donor states of a novel bacterial reaction center (Zn-RC) of Rhodobacter sphaeroides, containing six Zn-bacteriochlorophylls (Zn-BChls). A "Zn-β-RC" in which the Zn-BChl in the bacteriopheophytin (BPhe)-binding site on the A side (H(A)) has the Zn penta-coordinated, was also studied. The fifth ligand comes from a histidine introduced by site-directed mutagenesis. Formation of the P(+)Q(A)(-) state was observed in both types of RC, although under identical experimental conditions a significantly deeper P(-) band (corresponding to the lower-energy, special pair, excitonic component) was revealed in the Zn-RC. Assuming a similar lifetime of the P(+)Q(A)(-) state, the quantum yield of P(+)Q(A)(-) formation decreased by ~60% in the Zn-β-RC (compared to the Zn-RC), as was seen in a comparison of analogous (Mg) BChl-containing wild type and β-RCs of Rb. sphaeroides [Kirmaier et al. Science1991, 251, 922]. However, the average (weakly frequency-dependent) low-temperature electron transfer (ET) rates of the Zn-RC and Zn-β-RC (measured from zero phonon holes in resonant transient HB spectra) were both ~1 ps and similar to a rate previously measured in the Rb. sphaeroides native RC [Johnson et al. J. Phys. Chem. 1989, 93, 5953]. Electron transfer rates observed in this work on the Zn-RC yielded a P870* decay rate in good agreement with recent room-temperature, time-domain data [Lin et al. Proc. Natl. Acad. Sci. 2009, 106, 8537]. A lack of correlation observed between the holes near 810 and 883 nm, accounting for electrochromically induced shifts of the Zn-BChl transitions in the B(A,B) and H(A,B) binding sites, produced by formation of the P(+)BHQ(A)(-) state, indicates that the 810 nm bleach does not correspond to the P(+) (upper excitonic component of the dimer) band and is mostly contributed to by a shift of the B(B) absorption band. ZPH-action spectra indicated inhomogeneous broadening (Γ(inh)) of ~110 cm(-1) (Zn-RC) and ~130 cm(-1) (Zn-β-RC). Experimentally determined Γ(inh) decreased the number of variables in theoretical fits of the absorption and frequency-dependent shapes of resonant HB spectra, leading to more reliable Huang-Rhys factors for both low-frequency phonons and a pseudolocalized phonon, ω(SP), often referred to as the special pair marker mode.