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Unlike the phage described above, 29 replicates its genome as a monomer. This is accomplished through a protein-primed DNA replication mechanism, which yields individual genomes with a terminal protein covalently attached to the 5' ends of the duplex Fig. Genome packaging requires a "packaging ATPase" protein that associates with the terminal protein. This enzyme also binds to the 29 portal complex to complete the packaging motor. Despite the apparent difference, this mechanism is quite similar to the general packaging strategy, as follows.
The small terminase subunits described above provide specific recognition of viral DNA, while the large subunits possess the ATP-powered packaging activity. In the case of 29, the terminal protein 30 kDa is strictly required for genome packaging and may be viewed as a small terminase subunit. Further, the packaging ATPase of 29 is analogous to the large subunits found in the conventional terminase holoenzymes of X,, T4, etc.
The general strategy for genome packaging is thus retained in 4 Viral Genome Packaging Machines: Genetics, Structure, and Mechanism The majority of this book examines genome packaging in the dsDNA viruses. In reading these chapters, it becomes apparent that the basic mechanisms of energy transduction Unked to DNA translocation are quite similar.
This conceptual model is not limited to DNA packaging machines, however. The mechanism of genome packaging in 6, a double-stranded RNA virus, is reviewed in Chapter 8 of this book Poranen, Pirttimaa and Bamford. In a twist from the dsDNA viruses, this motor is also responsible for extrusion of newly synthesized message RNAs from the capsid upon the next round of infection. Importandy, the prokaryotic RNA packaging system shows functional similarity to the eukaryotic reoviruses, and again suggests that a general packaging mechanism traverses prokaryotic-eukaryotic boundaries.
A coherent mechanistic model for any complex biological process requires i a description of the macromolecules involved, ii a detailed understanding of how these molecules interact in the formation of larger biological structures, iii a description of the catalytic activities associated with these complexes, and iv an accounting of the processes that link catalytic activity to structure and function. Genome packaging is a crucial step in virus assembly in a number of prokaryotic and eukaryotic viruses. The molecular motors responsible for this process show mechanistic similarity in viruses as distinct as bacteriophage A.
The chapters in this book provide a detailed summary of our current state of knowledge of the genetics, biochemistry and structure of these fascinating motors. We hope that this book provides the experimental background and a philosophical roadmap towards this goal. In viruses as diverse as bacteriophage X and the herpesviruses, DNA replication proceeds through a rolling circle mechanism where the circular genome serves as a template for the synthesis of linear concatemers multiple genomes in length.
Concurrendy, viral gene expression produces structural proteins, which self-assemble into procapsids and, in the case of the bacteriophage, tails necessary to assemble an infectious virion. Virus assembly requires that monomeric virion DNA molecules be produced from concatemers during packaging of the DNA into a procapsid.
Thus, packaging represents the convergence of the DNA replication and capsid shell assembly pathways. Genome packaging in bacteriophage X has been extensively studied and this system has been used as a paradigm for virus assembly. Here we summarize current knowledge, present a working model, and indicate issues worthy of further investigation. The linear genome immediately circularizes via base, complementary "sticky" ends and the nicks are sealed by host ligase yielding a circular duplex. The annealed sticky ends form one subsite of cos, the cohesive end site of the X genome.
The decision of which pathway to enter depends on the physiology of the host cell and the multiplicity of infection. In the lysogenic pathway, lytic genes are repressed and the viral chromosome integrates into the host chromosome by site-specific recombination, forming a repressed prophage.
The second fate is the lytic pathway. In this case, the lambda O and P genes are expressed, yielding replication proteins that initiate viral DNA synthesis at ori. Initially, DNA synthesis by E. Later during infection, a rolling circle mechanism o replication predominates, which produces linear end-to-end polymers of X chromosomes, called concatemers. Circular concatemers are also produced by recombination between circular molecules, but linear concatemeric DNA is the major substrate for the assembly of infectious virions.
Developmental pathway for baaeriophage lambda.
Panel A: Infeaion of an E. Infeaion initiates with adsorption ofthe virus to the cell surface 1 followed by "injeaion" ofviral DNA into the cytosol of the host cell 2. The linear genome circularizes via the base single-stranded cohesive ends red dots and the nicks are sealed by host ligase 3. DNA replication initially proceeds via a bi-direaional 0 replication mechanism yielding daughter circles 4.
At later times, DNA is replicated via a rolling circle a mechanism that produces end-to-end concatemers ofthe viral genome 5. The duplicated cosskes in concatemeric DNA are indicated with red dots. Circular concatemers are also produced via recombination 6. Transcription and translation of the viral genome 7 yields structural proteins required to assemble an infectious virus. Panel B: Processing and packaging ofviral DNA into a procapsid, and tail attachment to yield an infectious virion. The terminase enzyme blue oval binds to a cos site in the concatemer 1.
Strand separation requires ATP hydrolysis. DNA packaging promotes capsid expansion, which increases the volume of the capsid and increase angularization of the capsid. The translocating terminase complex stops at the downstream cosskc and again nicks the duplex terminal c 8 Viral Genome Packaging Machines: Genetics, Structure, and Mechanism recruits a procapsid, sponsors insertion of the DNA into the procapsid, and finally cuts the end of the genome to complete the packaging process Fig.
As with other terminase enzymes, X, terminase is a heteroligomer composed of small gpNul and large gpA subunits see Fig. This is accomplished through a site-specific endonuclease activity that introduces nicks into cos that are staggered by 12 bp. GpA also has a so-called "helicase" activity that separates the nicked strands thus generating the single-stranded "sticky" ends of the mature genome. While the large terminase subunit possesses all of the catalytic activities required to cut and package the viral genome, gpA alone exhibits low catalytic activity.
The biological activities of X terminase are discussed in detail below. In summary, the packaging pathway entails terminase assembly at a cos site in the concatemer and cutting of the duplex the initial cos cleavage reaction , which yields the mature left end of the genome to be packaged. Upon binding a procapsid, the packaging machinery translocates DNA into the capsid through a capsid structure known as the portal vertex active DNA packaging. Upon arrival at the next downstream cos site in the concatemer, the packaging machinery stops and terminase again cuts the duplex generating the mature right end of the genome the terminal cos cleavage reaction ; this process yields a single viral genome tightly packaged within the confines of the capsid as described in Figure IB.
The site where terminase introduces staggered nicks to generate the cohesive ends is called cosN Fig. Early during the study of X, it was thought that cosN was both necessary and sufficient for DNA packaging. Later studies showed, however, that cos is complex and consists of three and perhaps four distinct subsites. Conversely, efficient termination requires the presence oicosQj a subsite that is located upstream of foW Fig.
The 12 sequence is located between cosN and cosB, and also plays a distinct role in efficient DNA packaging. Each of these subsites is discussed in detail below. Many of the base pairs bp within cosN show two-fold rotational symmetry, which extends over 22 bp if one includes purine-purine and pyrimidine-pyrimidine symmetry Fig. This argument is ftirther supported by i analogies to the interactions of type II restriction endonucleases with their palindromic recognition sequences, and ii the presence of a leucine-zipper motif in the primary sequence of gpA.
The cos region of a lambda concatemer. The cosB subsite is composed of the 11 and R-elements, as indicated. The 12 region lies between cosNsind the R3 element. The center of symmetry of cosN is indicated with a dot. Terminase normally nicks the duplex at Ni and N2 sites indicated with arrows. Lower panel: Strand separation by terminase yields the matured D R and DL ends of the lambda genome, as shown. T h e cosB subsite contains three 16 bp R elements that are specifically recognized by g p N u l Fig.
This II site introduces an intrinsic bend into the duplex, and is also specifically recognized by IHF. Moreover, packaging is not arrested in the absence of cosQ, and additional D N A , including the downstream cos, is packaged until the capsid shell is filled to capacity. Domain organization of the terminase gpNul and gpA subunits. Upper panel shows gpNul. Lower panel shows gpA. In both panels, sites covalently modified with 8-azido ATP are indicated with asterisks, and specific proteolysis sites are indicated with arrows, respectively.
Details are presented in the text. Rather, cosQ acts in concert with cosNsaid 12 to promote efficient termination. Here we refer to the region between cosN and cosB, namely bp 18 to 49, as the 12 subsite for historical reasons. Bacteriophage Lambda Terminase and the Mechanism of Viral DNA Packaging initiate a second round of packaging, and terminase thus packages successive genomes in the concatemer in a processive manner.
While the terminal roj-cleavage reaction requires only the 12 element, processive packaging requires that the cosB subsite also be present. X terminase is a heteromultimer of gpNul and gpA subunits. Here we discuss the structure, catalytic activities, and function of the individual subunits, and the holoenzyme complex. Ortega and C. Catalano, unpublished. Nucleotides do not affect the DNase protection pattern, however. Gaussier and C. The capsid genes of phages X and 21 descend from a common ancestor and are of similar size, function and genetic structure.
Despite sequence homology, the X and 21 gene products are not interchangeable due to divergent interaction specificities. In other words, X terminase packages X DNA specifically into X procapsids, while phage 21 terminase specifically utilizes phage 21 procapsids. The C-terminal 40 amino acids of the protein are required for efficient gpA-binding interactions and holoenzyme formation. Residues LyslOO - Pro define a hydrophobic domain that is required for high-afFinity DNA binding interactions; deletion of this self-association domain decreases DNA binding interactions by three orders of magnitude.
It has been proposed that this helix forms a flexible linker between the two domains that alternately plays a role in i cooperative gpNul binding interactions at cosB and ii cooperative assembly of gpA at the roWsubsite. The average of the 20 lowest energy structures obtained from NMR is shown in ribbon representation. The gpNul dimer blue binds to two R-elements R3 and R2 spatially separated in the duplex. Note that wing residues are positioned to directly interact with bases in the minor groove of the duplex.
Reproduced from reference 43, with permission. Importantiy, this model accommodates the observed effects of IHF on lambda development, which are detailed below. IHF modulates A. In this model, IHF binding to II introduces a strong bend in the duplex, which juxtaposes the R3 and R2 gpNul binding sites in the appropriate orientation. A single gpNul dimer spans these two "half-sites" that are separated by 44 bp in the duplex. We recognize, however, that a X mutant unable to utilize IHF would be at a disadvantage in the environment. Mutation of an R-element decreases intrinsic gpNul binding affinity, which places increased emphasis on the bend; IHF binding provides the prerequisite DNA structure.
While this model is fully consistent with all of the genetic, biochemical, and structural data available, it does not direcdy address the role of the Rl-element in gpNul assembly. Of note, howeyer, is that this element is dispensable in the presence of IHF in culture. Other domains are described in detail below. The data are consistent with genetic experiments demonstrating that both nuclease and helicase activities localize to the C-terminal half of gpA, and further suggest that there is a structural and functional overlap between the two catalytic activities.
We note that the endonuclease and helicase activities are stimulated and fueled, respectively, by ATP. J Mol Bio a; The Self-Association Domain s It is presumed that rotationally symmetric gpA subunits bound to cosNsire responsible for duplex nicking based, in part, on the two-fold rotational symmetry of the cosN sequence see Fig. Mutation of either Tyr46 or Lys84 strongly affects both ATPase and DNA packaging activities of terminase holoenzyme, but does not significantly affect cos cleavage or helicase activities. All of these motifs are found in the primary sequence of the X terminase gpA subunit.
Interestingly, the two mutations that abrogate the ATPase and DNA packaging activities of X terminase discussed above correspond to conserved motifs found in all of the terminase large subunits: i Tyr46 is a strictly conserved residue found in the adenine binding motif and ii Lys84 immediately follows a conserved Walker A motif found in gpA.
Rao and coworkers have demonstrated that mutation of an N-terminal Walker A sequence of the large subunit of bacteriophage T4 terminase abrogates the ATPase and DNA packaging activities of enzyme, without loss of DNA cutting functions. Closer inspection reveals that gpA possesses a second, albeit weaker, match to the P-loop consensus sequence in the N-terminus of the protein, positioned at Lys The holoenzyme also possesses multiple ATPase catalytic sites that play unique roles along the packaging pathway.
Each of these catalytic activities is discussed in turn below. Nuclease Activity While the isolated gpA subunit will cut a cos containing DNA substrate, this nuclease activity is strongly stimulated by interactions with gpNul. ATP modulates the nuclease activity of the holoenzyme in two important ways. First, ATP strongly stimulates the rate of duplex nicking.
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A second effect of ATP is the modulation of nuclease fidelity; in the absence of ATP terminase incorrecdy nicks the duplex, predominandy generating a four base pair nick which is lethal to virus development Fig. Introduction of mutations into the right cosN half site cosNR, Fig. Conversely, introduction of a symmetric mutation into the left cosN half site cosNL has dramatic effects, significantly decreasing the rate of the cos cleavage reaction in vitro and virus yield in vivo.
Introduction of secondary mutations in cosBy or complete deletion of cosB removes this asymmetry, and cosNR and cosNL mutations have identical effects. These data suggest that intrinsic DNA binding interactions between gpA and cosNL are critical to efficient duplex nicking, and that introduction of mutations into this DNA site strongly affect the reaction. Conversely, gpA binding to the cosNR half site is supported by cooperative interactions with gpNul assembled at cosB Fig.
Model for cooperative assembly of gpA red rectangle and gpNul blue rectangle at cos. A symmetric gpA dimer binds to cosN. Binding ofgpA to the cosNL half-site relies stricdy on intrinsic binding interactions. Strand-Separation Helicase Activity Subsequent to duplex nicking, the annealed strands are actively separated by the holoenzyme Fig. While this reaction has been described as a helicase activity, processive duplex separation has not been demonstrated.
IB and 2. Early studies isolated a stable intermediate in the packaging reaction in vivo. This species, known as complex I, could be isolated on sucrose gradients and chased into infectious virus with the addition of a cell extract containing X tails. The stability of complex I, presumably mediated by gpNul interactions with cosB, is likely critical for protection of the matured genome end prior to DNA packaging in vivo; should the complex prematurely dissociate, the 12 base single-stranded end is expected to be rapidly degraded by cellular nucleases, a lethal event.
The nature of complex I is more fidly described below. Subsequent mutational analysis indeed confirmed that the high- and low-affinity ATPase sites were located in the gpA and gpNul subunits, respectively. Mutational analysis yielded conflicting data, however. Mutation of the "critical" P-loop lysine in gpNul Lys35 indeed abrogated ATP hydrolysis by this subunit, but only when limiting concentrations of DNA were used; increasing the concentration of DNA in the reaction mixture restored the ATPase activity to wild type levels.
The gpA ATPase Catalytic Sites As discussed above, a P-loop motif was also identified in the gpA subunit; however, mutation of the "critical" lysine in the putative P-loop Lys had only minor effects on the observed rate of ATP hydrolysis by this subunit. Clues to the location of the high-affinity ATPase catalytic site were provided by affinity labeling studies using photoreactive ATP analogs.
Mutational analysis of these residues confirmed that both are required for high-affinity ATPase activity. Moreover, these residues are intimately involved in DNA packag75 77 mg activity. Summary of allosteric interaction between the catalytic sites of terminase holoenzyme. The catalytic activity of the isolated gpA red rectangle and gpNul blue sphere subunits is shown at top. Assembly into the holoenzyme activates catalytic activity as indicated in step ii. Interaction between the multiple catalytic sites of the holoenzyme is shown in the lower half of the figure.
Details are described in the text. S u n u n a i y o f t h e Allosteric Interactions b e t w e e n Terminase Catalytic Sites Allosteric interactions between the multiple catalytic sites of terminase holoenzyme have been observed, and are summarized in Figure 6. It is likely that these interactions are central to the assembly of the packaging machinery onto viral DNA, to promoting stability of intermediate packaging complexes, and to modulating the procapsid-dependent transition to a mobile packaging machine. These concepts are incorporated into a model for terminase assembly into a DNA packaging machine, described below.
Procapsid Assembly The X procapsid, also known as the prohead, is an icosahedron composed primarily of gpE, the major capsid protein. The portal also serves to nucleate capsid assembly, and it is likely that portal protein s are an active part of the DNA packaging motor. Procapsid assembly is a step-wise process that, in addition to gpE, requires the phage proteins gpB, gpC and gpNu3, and host groELS chaperonins. The process begins with the oligomerization of gpB monomers into a preconnector, a 25 S dodecameric ring with a A hole at its center Fig.
Procapsid assembly and processing. The phage-encoded gpC protein next adds to the preconnector to yield a 30S initiator structure; this reaction may also involve gpNu3. The initiator serves to nucleate copolymerization of gpNu3 and gpE, which yields an immature procapsid Fig. GpNu3 serves as a typical scaffolding protein, directing the polymerization of gpE into an icosahedral shell structure. Processing of the X Portal The X portal, unlike the simple portals found in other viruses, is quite complex both in terms of protein composition and protein modification. A second step in processing of the X portal is formation of the pXl and pX2 proteins referred to collectively as pX proteins.
Remarkably, these proteins appear to derive from an uncharacterized covalent fixsion product of gpC and gpE proteins. The chemical nature of these proteins and the mechanism of their formation remain completely unknown. The temporal relationship between gpB proteolysis, cross-linking of gpC and gpE, proteolysis of pY to yield pXl and pX2, and gpE assembly into a procapsid is unclear. The final step in procapsid maturation is proteolysis of the gpNu3 scaffolding protein and exit of the products from the procapsid Fig.
The initiation of DNA packaging in X is considered to include i assembly of the packaging machinery at a cos site in the concatemer, ii duplex nicking at cosN, and iii strand separation to yield complex I Fig. DNA translocation into the capsid defines propagation, which includes those steps that i promote the transition from the exceptionally stable complex I to a mobile packaging motor, and ii active translocation of DNA into the capsid Fig. Finally, termination includes those events responsible for i recognition of the terminal cos sequence by the translocating complex, ii duplex nicking and strand separation to release the DNA-filled capsid from the terminase-concatemer complex, and iii addition of accessory proteins and a tail to yield an infectious virus Fig.
Each of these processes is discussed in turn below. As discussed above, the role of Rl in this initial assembly is unclear. Whatever the case, gpNul bound at cosB serves to anchor a symmetric gpA assembly at the roWhalf-sites yielding a prenicking complex Fig. We presume that a gpA dimer assembles at cosN, but the structural details and stoichiometry of the subunits assembled at cos remain speculative. The presence of a bZIP protein dimerization motif in gpA suggests that an even number of subunits is involved, however.
ATP modulates the assembly and stability of the terminase subunits bound at cos as follows. Initiation of DNA Packaging. Initiation steps include terminase assembly at a cos site in the concatemer and maturation of the DL end. The terminase gpA and gpNul subunits are depicted as red ovals and blue spheres, respectively. IHF is shown as a purple lobe. Separation of strands by the so-called "helicase" activity of gpA leads to release of the right cohesive end DR from the complex Fig.
The products of the helicase reaction have not been characterized in detail and it is interesting to consider the fate of the terminase proteins bound at cos upon strand separation. Conversely, it is feasible that separation of the cohesive ends requires remodeling of gpA assembled at the two roW half-sites. The two obvious outcomes are that i gpA bound to the cosNL half-site separates from the complex along with the D R fragment as shown in Fig.
While the structural details of this transition must await further biochemical analysis, we propose that strand separation ultimately yields two products in which terminase proteins remain bound at each chromosomal end. This is discussed further below. What Is Complex U This stable intermediate was originally described in vivo, and was defined as terminase bound to concatemeric DNA at uncut cos sites. We further propose that "complex F' formed in vivo is in fact identical to the stable complex that we have characterized in vitro; specifically, terminase tighdy bound to the D L fragment formed by duplex nicking and strand separation, as shown in Figure 8.
Stoichiometric r6? The ensemble of data fully support a model where stoichiometric duplex nicking and strand separation by terminase in vivo yields a stable complex in a manner identical to what is observed in vitro; we refer to this intermediate as complex I. It is likely that the stability of this complex is required to protect the single-stranded left cohesive end DL from nuclease damage in vivo, though this has not been direcdy demonstrated.
Conversely, the D R complex is relatively unstable, and dissociation of the terminase protein s leads to degradation of the right DNA end by host nucleases Fig. This series of events is likely initiated by interactions of complex I with the portal proteins in the procapsid, and is also modulated by the phage gpFI protein, as discussed in detail below. The next step in the packaging pathway is the transition to a translocation complex, a reaction that is also mediated in an ill-characterized manner by the phage gpFI protein. One model invokes a negative regulation mechanism where unassembled capsid proteins directly inhibit the endonuclease activity of terminase; assembly of these proteins into a procapsid effectively removes the inhibitor s from solution, relieving the repression oicos cleavage.
Translocation of DNA into the Procapsid. The procapsid is shown as a large cyan sphere containing a portal complex purple oval. This model suggests that terminase binds cos and is poised to nick the duplex, but requires gpFI and procapsids to stimulate the reaction. The in vivo cos cleavage results described above are in direct contrast to in vitro studies showing that terminase can efficiently cut ro5-containing DNA in the absence of gpFI and 26 Viral Genome Packaging Machines: Genetics, Structure, and Mechanism procapsids.
Thus, neither procapsids nor gpFI affect duplex nicking by terminase in vitro, and we propose that terminase may similarly cut cos in the absence of these factors in vivo see the discussion of complex I above. A Reversibility Model for the Procapsid Requirement for cos-Cleavage in Vivo If terminase does not need to be activated to cut coSy how might the in vivo requirement for procapsids be explained.
This predias that during an infection by a mutant phage unable to produce procapsids, duplex nicking and strand separation should proceed normally. A recendy proposed reversibility model accounts for the apparent procapsid dependence of terminase in vivo. For concatemer processing by these phages, an initial cut is made at a defined site called pac.
DNA cutting may be irreversible when the concatemer cleavage products lack cohesive ends. Model for gpFI Requirement in Vivo As described above, phage with mutations in the FIgene also exhibit an apparent lack of c 28 Viral Genome Packaging Machines: Genetics, Structure, and Mechanism proposed that the Efi domain is located on the surface of the procapsid and that complex I direcdy interacts with the Efi domain en route to docking at the portal.
In this model, gpFI direcdy modulates this initial interaction and guides the procapsid-terminase docking step. In this model, the Efi domain represents a portal docking site that makes direct contacts with terminase in complex I. The finB mutations presumably allow this interaction in the absence of gpFI. Both of these proteins bind to specific sites in cosB, and are likely central to the stability of complex I. It thus clear that a major alteration in protein-DNA binding interactions must take place prior to translocation.
IHF most likely dissociates from complex I to allow passage of gpA and the procapsid, as shown in Figure 9. Indeed, based on the effect of gpFI on the roj-cleavage reaction in vitro, we have suggested that gpFI may act antagonistically to IHF in modulating the stability of complex I. One possibility is that gpNul switches from a site-specific DNA binding protein to a nonspecific DNA binding protein that is an active part of the packaging motor shown in Fig.
This model is consistent with the observation that small terminase subunits from bacteriophage T4 and SPPl form oligomeric rings in solution, which may indicate their role as a "sliding clamp" during translocation. A final possibility is that gpNul is ejected from the translocating complex. This model suggests that the role of gpNul is to site-specifically assemble gpA at cosN, and maintain the integrity of complex I until the procapsid arrives.
Thus, gpNul plays a role analogous to that of sigma factors and transcription factors in the assembly of the transcription complex. Once the appropriate nucleoprotein complex has been assembled, movement of the protein machinery results in ejection of the assembly protein s from the DNA. Again, the answers to these questions must await further experimentation. Consistently, sequence analysis of terminase enzymes from phage to the herpesviruses indicates the presence of conserved ATPase motifs in these proteins.
The observed stoichiometry in the X system is two ATP s consumed per base pair packaged, though this may represent a significant overestimation. This is significantly greater than the basal rate of 50 min' for gpA in the holoenzyme. Models for DNA Translocation Once released from the cos site, the packaging machinery translocates along the duplex, actively inserting DNA into the capsid.
How might such a translocation machine work? The goal of understanding a biological motor at the molecular level challenges us to mechanistically link the energy of ATP hydrolysis to physical changes in protein structure that lead to translocation. Significant progress has been made towards our understanding of a number of motors, including myosins, kinesins, and the rotary FIFQ and flagellar motors. It is likely that both terminase proteins and portal proteins assemble to complete a translocating motor, but the exact nature of the complex is unknown in all cases, ii Once assembled, the motors act quickly and transiently.
Dissociation of the components occurs upon completion of the packaging process, iii There is little structural information available for any of the components of these machines, and none on the actively packaging motors. Nevertheless, it is clear that there are three parts to the motor: terminase, the portal vertex, and the procapsid shell. There are a number of creative models describing how the components of the packaging complex sponsor DNA movement. The first proposes that the terminase enzyme is direcdy responsible for translocation of DNA into the capsid. These models propose that the terminase subunits physically translocate via flexible, DNA-contacting domains that cyclically contract, and then undock from the DNA.
These models find mechanistic similarity to the "inch worm" mechanism proposed for translocation of hexameric helicases. In one version, which is actually the earliest model, ATP hydrolysis drives rotation of the portal protein with respect to the capsid shell, which "screws" the DNA into the capsid. A more recent portal model is inspired by the 30 Viral Genome Packaging Machines: Genetics, Structure, and Mechanism structure of the t 29 portal Anderson and Grimes, Chapter 7 of this work.
This model proposes that the procapsid-terminase complex which possesses ATPase activity acts as a stator, the DNA as a spindle, and the portal complex as a ball-race. The strongest evidence comes from genetic studies in which lethal mutations in the virus were screened to identify those that affected DNA packaging. These mutants provide evidence for the involvement of the terminase gpA subunit in translocation and active DNA packaging.
A detailed biochemical analysis of these mutants in vitro will undoubtedly provide fixrther insight into the role of gpA in the packaging motor. The viral gpD protein, which is a monomer in solution, adds to the surface of the expanded icosahedral capsid as a trimer, and in numbers similar to the major capsid protein Fig. The intermediate stability of X shp virions indicates that the shp protein imparts considerable stability to the capsid shell, even though the shell is not completely populated by Shp. When terminase encounters this site, translocation stops and symmetric nicks are introduced into the cosNsuhske Fig.
The strand separation activity of terminase separates the cohesive ends, releasing the DNA-fiUed capsid from the concatemer. Terminase remains bound to the matured D J end of the concatemer, which represents the next X chromosome to be packaged. The complex, which is functionally-related to complex I, can bind a new procapsid and initiate packaging of the second chromosome in the concatemer.
Terminase-mediated genome packaging is thus processive, and it has been estimated that genomes are packaged per DNA binding event.
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Termination of DNA Packaging. Termination requires recognition ofcosNhy the translocating complex. The cosQ-cosN-l2 segment is central to capture of the packaging machinery. Duplex nicking and strand separation complete DNA packaging. Addition of gpW, gpFII and a tail complete the assembly of an infectious virus.
Our interpretation of the bottom strand nicking failure of A, cosCl is based on symmetry issues, as follows. We presume that the gpA oligomer hexamer? We propose that cosCl plays a direct role in this reorganization, and is required to sponsor the presentation of a properly oriented terminase complex to the cosN half-sites. When the genome is shortened to lengths Processive Genome Packaging Following the downstream cos cleavage event, the terminase proteins dissociate from the portal complex of the DNA-fiUed capsid, but remain bound to the matured DL end of the next genome in the concatemer Fig.
This terminase-DNA complex, which is functionally related to complex I, captures a procapsid to sponsor the packaging of the next chromosome in the concatemer. While the cosB subsite is not essential for the terminal cos cleavage reaction, it is required for processive genome packaging. These data indicate that the terminase-DNA interactions required for Bacteriophage Lambda Terminase and the Mechanism of Viral DNA Packaging 33 processive packaging are "relaxed" in comparison with the requirements for the initial assembly of terminase at cos see packaging initiation, above.
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Structural studies on these complexes are needed to understand the DNA site requirements for each of the packaging complexes. Virion Completion Subsequent to separation of the DNA-filled capsid from the concatemer, the minor capsid proteins gpW and gpFH are sequentially added to the portal Fig. The solution structure represents a novel fold, consisting of two a-helixes and a two-stranded P-sheet, arranged around a hydrophobic core.
The 14 C-terminal residues, which are essential to virus development, are disordered in the structure. This structure is composed of seven p-strands and a short a-helix, with two unstructured regions extending between residues and GpFII has homologues in other lambda-like phages, such as 21 and 80, and it is possible to compare sequence alignments with binding specificity.
Of particular interest is the gpFII analogue from 80, because this protein is tail specific. That is, gpFII 1 80 forms infectious virions with 80 tails, but not with X tails. Concluding Remarks Assembly of an infectious X virus starts with the products of the DNA replication and procapsid assembly pathways. Addition of the "finishing proteins" gpW and gpFII, followed by tail attachment complete the infectious virion. An ordered and essentially irreversible series of macromolecular assembly steps are required to carry out the interdependent processes of i cutting concatemeric DNA into unit-length virion chromosomes, ii packaging the chromosomes into procapsid shells and iii stabilization of the DNA-filled capsid and tail attachment.
Further, high-resolution structures of several assembly proteins, including gpD, gpW, gpFII and the DNA binding domain of gpNul have been determined, providing a glimpse into the structure-ftinction relationships of these critical proteins. As an experimental system, X is highly developed, with excellent genetics and strong biochemistry. Each of the proteins involved in X assembly has been cloned and purified which sets the stage for a detailed characterization of virus development at the molecular level.
We are thus challenged to characterize the biochemical, structural and functional aspects of each step along the developmental pathway leading to an infectious virus. These studies remain a challenging area of research, but will undoubtedly lead to significant insight into the molecular mechanisms of virus assembly.
We further wish to acknowledge the seminal work in the Toronto labs of Andy Becker, Marvin Gold and Helios Murialdo, which forms the basis for many of the models presented in this review. References 1. Katsura I. Tail assembly and injection. Lambda II. Herskowitz I, Hagen D. The lysis-lysogeny decision of phage lambda: Explicit programming and responsiveness. Ann Rev Genetics ; Ptashne M, Gann A. Friedman D, Gottesman M. Lytic mode of lambda development. Cell ; 54 6 The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A.
The relationship with their DNA helicase and packaging activities. J Biol Chem ; 18 The in vitro translocase activity of lambda terminase and its subunits. Kinetic and biochemical analysis. J Biol Chem ; 34 Woods L, Catalano C. Kinetic characterization of the GTPase activity of phage lambda terminase: Evidence for communication between the two "NTPase" catalytic sites of the enzyme. Biochemistry ; Kinetic analysis of the endonuclease activity of phage lambda terminase: Assembly of a catalytically competent nicking complex is rate-limiting.
Biochemistry ; 36 19 Cue D, Feiss M. J Mol Biol ; Separate sites for binding and nicking of bacteriophage lambda DNA by terminase. Structure of the bacteriophage lambda cohesive end site: Location of the sites of terminase binding cosB and nicking cosN. Gene ; 24 Hohn B. Miwa T, Matsubara K. Lambda phage DNA sequences affecting the packaging process.
Gene ; A site required for termination of packaging of the phage lambda chromosome. Davidson A, Gold M. Mutations abolishing the endonuclease activity of bacteriophage X terminase lie in two distinct regions of the A gene, one of which may encode a leucine zipper DNA binding domain. Virology ; The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction. J Mol Biol a; 3 Higgins RR, Becker A.
EMBO J a; 13 24 Xin W, Feiss M. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB. Yeo A, Feiss M. Specific interaction of terminase, the DNA packaging enzyme of bacteriophage lambda, with the portal protein of the prohead. J Mol Biol ; 2 : Cue D , Feiss M.
Genetic evidence that recognition of cosQ, the signal for termination of phage A, D N A packaging, depends on the extent of head filHng. Genetics ; Alterations of the portal protein of bacteriophage X suppress mutations in cosQ, the site required for termination of D N A packaging. Submitted Genetics ; Wieczorek D , Feiss M. Defining cosQ, the site required for termination of bacteriophage lambda D N A packaging. Genetics 2 0 0 1 ; Genetics ; In press. Termination of packaging of the bacteriophage lambda chromosome: c o s Q is required for nicking the bottom strand of cosN.
J Mol Biol ; 1 : Miller G, Feiss M. J Virol 85 : — Viral DNA polymerases.
In DNA replication in eukaryotic cells ed. DePamphilis ML , pp.
Google Scholar. Antiherpesviral DNA polymerase inhibitors. In Antiviral research: Strategies in antiviral drug discovery ed. LaFemina R , pp. Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc Natl Acad Sci 88 : — Apoptosis 16 : — ND10 components relocate to sites associated with herpes simplex virus type 1 nucleoprotein complexes during virus infection.
J Virol 78 : — Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome. Proc Natl Acad Sci 97 : — Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis. J Virol 82 : 96 — Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function. Science : — Construction and properties of a recombinant herpes simplex virus 1 lacking both S-component origins of DNA synthesis. J Virol 67 : — Phosphorylation of the herpes sVirus type 1 origin binding protein.
J Virol 75 : — Mutations that increase DNA binding by the processivity factor of herpes simplex virus affect virus production and DNA replication fidelity. Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2. Human paillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase. Herpesvirus replication compartments originate with single incoming viral genomes.
Proliferating cell nuclear antigen uses two distinct modes to move along DNA. Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion. Structure 16 : — Medline Google Scholar. Crystal structure of the herpes simplex virus 1 DNA polymerase. Molecular chaperones and alphaherpesvirus infection. In Alphaherpesviruses: Molecular virology ed. Weller SK , Chap. Caister Academic , Norfolk, UK. Oligomerization of ICP4 and rearrangement of heat shock proteins may be important for herpes simplex virus type 1 prereplicative site formation.
J Virol 82 : — Recombination-dependent concatemeric viral DNA replication. Virus Res : 1 — Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication. J Biochem : — J Cell Sci : — The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures. EMBO J 15 : — J Mol Biol : — A dimer of Escherichia coli UvrD is the active form of the helicase in vitro.
Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication. The residue C-terminal region of the single-stranded DNA binding protein of herpes simplex virus type 1 is required for cooperative DNA binding. J Virol 74 : — The crystal structure of the herpes simplex virus 1 ssDNA-binding protein suggests the structural basis for flexible, cooperative single-stranded DNA binding.
Existence of transdominant and potentiating mutants of UL9, the herpes simplex virus type 1 origin-binding protein, suggests that levels of UL9 protein may be regulated during infection. Helicase motif Ia is involved in single-strand DNA-binding and helicase activities of the herpes simplex virus type 1 origin-binding protein, UL9.
J Gen Virol 77 : — Maribavir prophylaxis for prevention of cytomegalovirus disease in recipients of allogeneic stem-cell transplants: A phase 3, double-blind, placebo-controlled, randomised trial. Lancet Infect Dis 11 : — DNA mismatch repair proteins are required for efficient herpes simplex virus 1 replication. PCNA, the maestro of the replication fork. Cell : — J Gen Virol 70 : — Replication and recombination of herpes simplex virus DNA. Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro. Stepwise evolution of the herpes simplex virus origin binding protein and origin of replication.
Herpes simplex virus type 1 immediate-early protein vmw induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase. J Virol 73 : — Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.
J Virol 61 : — Progress in the development of new therapies for herpesvirus infections. Curr Opin Virol 1 : — Human cytomegalovirus UL97 kinase is required for the hyperphosphorylation of retinoblastoma protein and inhibits the formation of nuclear aggresomes. Evidence that the herpes simplex virus type 1 uracil DNA glycosylase is required for efficient viral replication and latency in the murine nervous system. J Virol 68 : — Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis.
PLoS Pathog under revision. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates. J Gen Virol 91 : — Nucleolin associates with the human cytomegalovirus DNA polymerase accessory subunit UL44 and is necessary for efficient viral replication.
Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments. Nat Struct Mol Biol 16 : — Bacteriophage-encoded functions engaged in initiation of homologous recombination events. Crit Rev Microbiol 35 : — Assembly of an active translation initiation factor complex by a viral protein. Genes Dev 20 : — Herpes simplex virus reorganizes the cellular DNA repair and protein quality control machinery.
One was new gene-editing technologies—ways of doctoring existing tickertape a letter at a time. This has breathed new life into the idea of making precise changes to genomes, which is what synthetic biology is all about. The falling cost of DNA synthesis, on the other hand, was widely foreseen.
But it has still been a dramatic enabler. The price of a gene synthesised to order is about a thousandth of what it was in ; if you buy in bulk or have the technology in-house it can cost a lot less. And then there is machine learning. Synthetic biology gets even greater benefits than most other industries from the recent growth in the capabilities of pattern-recognition programs. It is not just that laboratories produce reams of data with which to train such programs.
For machines, though, understanding is as unnecessary as it is impossible. They just find patterns and uncover rules. This is not science as scientists understand it. But, if rigorously tested, such rules can still be a basis for engineering. There were perfectly good rules for building bridges long before there was a theory of gravity. While synthetic biology has grown more capable, the promise of two older approaches to the improvement of life has diminished.
It was ten in It is well under one today, and still dropping. This has excited interest in fundamentally new approaches to medicine. One is reprogramming cells to do helpful therapeutic things. Immune-system cells are the most obvious candidates. The cells of the microbiome—the interlinked bacterial ecosystems that thrive on skin and in guts—are another possibility. The second ailing improver of life is the petrochemical industry. The mistake was rushing into a bulk market with low margins: petrol.
Some companies are now using synthetic biology to replace more upmarket molecules from the same crude oil which end up in fragrances and food additives with far more added value. Others are looking at making plastics environmentally friendly. As their technologies prove themselves at increasing scales, and as their technical prowess allows them to expand their repertoire to cheaper bulk products, these efforts could eat the petrochemical industry from within like some world-saving parasitic wasp.
Synthetic-biology executives say their worry is not money, but focus and time. Every firm has more revolutionary-looking projects than it can pursue. And no one knows how long it will take the projects to pay off. Proteins, which carry out almost all the basic functions of life, from respiration to reproduction, are all made of 20 smaller molecules strung together into a chain. The shapes those chains fold up into in order to fulfil different functions are complex and incredibly hard to predict.
But they are almost all entirely determined by the order of these smaller molecules, which are called amino acids. The gene for a given protein is simply the list, in order, of the amino acids needed to make it. This information is written down in the genome as a sequence of DNA bases— A , C , T and G , the letters on the ticker-tape—in the same way data in a computer are stored as a string of 1s and 0s.
The program that turns these DNA sequences into sequences of amino acids is the genetic code. Imagine a codebook with codons in one column and the names of the amino acids in another.
Functions and Utility of Alu Jumping Genes | Learn Science at Scitable
To decode a gene, look up its codons one by one and write down their amino-acid meanings. It is a simple, rule-based undertaking—an algorithm. The cell carries out the same algorithm. But instead of a code book which matches codons to amino acids, it uses codon-recognising, amino-acid-carrying molecules called t RNA s and a mechanism called a ribosome which provides a place for those t RNA s to interact with a copy of the gene.
The act of reading the gene, codon by codon, is the act of creating the protein, amino acid by amino acid see diagram. When it executes an algorithm this way, biology looks like computer science. But it is important to appreciate that biology does not deal with information the way humans do. In human programs, the logic and the machinery that acts on it are kept separate. Computer users can change a program in blithe ignorance of the physical principles and peculiarities built into the hardware that it runs on. But evolution cares nothing for such distinctions.
All its processing is just a matter of molecules interacting—the way that t RNA s stick to codons as if to velcro, the way the shape of the ribosome forces amino-acids together, and so on. From the simple gene-to-protein translation of the ribosome to the extraordinary synchronised symphony which turns a fertilised egg into a whole human, biological information and its implementation are all but inseparable. Life runs not on software and hardware, but in allware. That makes it highly resistant to human reprogramming.
It can, though, be hacked. From the 51 amino acids of human insulin, which in became the first product made by the first biotech company, Genentech, to artificial antibodies containing more than a thousand of the things, biotechnology consists almost entirely of getting cells to produce proteins they would not normally make by cutting a gene out of one organism and dumping it, often unceremoniously, into another.
Most of these proteins have been natural ones. Nature is well stocked with proteins that do useful things—regulate blood sugar, kill pests or break down grime on laundry. Putting the genes for such proteins into the genomes of bacteria that will then secrete insulin, or of crops that need pest resistance, or fermentation tanks churning out supplements for detergent, was an obvious moneymaker.
But the preference for the natural was, until recently, also driven by necessity. Designing a protein from scratch was impossibly hard. So was getting suites of proteins from different sources to work together. That is no longer true. Protein design and DNA synthesis now make it possible to produce proteins that, separately or together, do things nature does not. They remain imperfect. But because DNA sequences are cheap it is possible to try out lots of variations to see which actually work. Thus, for example, there are companies working on new metabolic pathways which combine enzymes freshly discovered through the sequencing of ever more genomes, enzymes long understood and enzymes significantly re-engineered.
It is an exacting craft, or art; it requires not just finding the right enzymes but also bringing about the carefully balanced levels of gene expression needed if a dozen or more of them are to work together, not to mention tweaking the underlying metabolism to prevent things produced by the new pathway from disrupting those already there. But if the work is done well, it seems now to be the case that more or less any small molecule found in nature can be made by yeast or bacteria in a fermentation tank.
Two particularly interesting possibilities are the cannabinoids made by marijuana and the variations on opium and morphine made by poppies. Cannabinoids come in a remarkably wide number of forms, some psychoactive, some not, some therapeutic, some not, many legal for some purposes in some jurisdictions, many illegal for all purposes elsewhere. A set of cannabinoid-synthesising pathways described by Dr Keasling and colleagues this February offers therapeutic and recreational possibilities along these lines which will be explored by a new company called Demetrix. Dr Smolke has founded a company, Antheia, which aims to use her new know-how to make opiates that are cheaper and so more accessible to the tens of millions around the world unable to get pain relief, and also to make opiates that are less addictive.
A more radical possibility, at least in terms of chemistry, than remaking and improving natural compounds is to create enzymes to catalyse chemical reactions nature never carries out. Take the task of sticking a carbon atom to a silicon atom.
Human chemists are pretty good at this, and the organo-silicon compounds they thus create are used in electronics, pharmaceuticals, building materials, breast implants and more. Nature, though, does not use carbon-silicon bonds, and so no natural enzymes make them. As well as making new proteins, it is also possible to make new RNA s. A molecule of RNA is created that velcroes itself to a specific sequence in the genome; a companion protein then slices through the bit of DNA thus highlighted. Once the DNA is broken, a new gene, or gene fragment, can be inserted into the gap.
If you put a gene describing the CRISPR RNA and its protein into a cell in such a way that it gets expressed only under certain conditions, you have a cell whose genome can be reprogammed by remote control. To this end they are carefully stitching together the most appropriate versions of over 6, genes as well as most of the sometimes vital gubbins found between them—over 12m bases of DNA in all. One of the things the project is writing into the genome is a system that will make it cut itself up and reshuffle its genes when told to.
This technology should provide a powerful new tool for the study of evolution, says Tom Ellis of Imperial College, London. Wherever the natural, baseline yeast genome marks the end of a protein-coding sequence with a TAG codon, the scientists writing Sc2. This means that in Sc2. Nature uses 20 amino acids in its proteins. But there are hundreds of others that could be used, some of which would confer interesting new properties. In Sc2. Cells thus equipped will be able to use an amino acid no natural cell has ever used before.
Nor does the process have to stop there. Rewrite the code with fewer synonyms, and you have more codons to devote to non-canonical amino acids. One therapeutic option this might open up is drugs that bacterial defences cannot cope with. Bacteria have evolved to counter everyday proteins; put in amino acids they have never seen before and some of those defences no longer work.
Bespoke genetic codes have attractions beyond a larger vocabulary. Recoding could thus make cells immune to any viral attack; indeed, there is already work on achieving this in bacteria. If it works, this sort of recoding could be very helpful to existing biotechnology. Fermentation tanks that never get wiped out by infections and antibody-producing cell lines that could not harbour viruses would be a great boon. It is possible to imagine changes in the way codons code for amino acids so radical that parts of synthetic biology become a separate creation, parallel biospheres based on the original but no longer in contact with it, populated by creatures which neither infect nor are infected, that are linked to the rest of life only through the intentionality of design.
A hint of such strangeness could be seen in a paper published in Science , a journal, this January by Stephen Benner of the Foundation for Applied Molecular Evolution in Florida and his colleagues. With eight letters to play with, for example, you could recode the genome to use doublets, rather than triplets, as codons, if you redesigned the ribosome, the t RNA s and a bunch of other stuff, too. Would anyone want to? The potential of the existing code is enormous, the range of proteins it can, in principle, describe is barely yet explored; there might seem to be no need for such showing off.
At the same time, engineers do like to tinker. Seeing brilliant metabolic engineering fail to make a business led him and his co-founders at Zymergen, a company based in Emeryville, California, to take their new company in a different direction. They would not try to manufacture or sell things. They would offer their synthetic biology as a way of making businesses already using biotechnology more profitable. This is, at the moment, the model used by a number of leading synthetic-biology companies.
At its heart is the automation of experiment. Biotechnology is already a bigger business than many people realise. The contribution was split between three industries. Chemicals used for many purposes—raw materials for plastics, food additives, some fragrances and biofuels—are already being churned out at scale by altered micro-organisms in fermentation tanks. As well as being the biggest biotechnology market, this is also the one best suited to companies seeking to offer innovation as a service.
Testing drugs and genetically modified crops is a long and costly business. Replacing one strain of industrial yeast with a better one can be done in a week. Industrial customers tend to know what they want and synthetic biology promises a lot of value. Tim Fell, the boss of Synthace, a synthetic-biology software company in London, says that in one project the company engineered a fold increase in the rate at which bacteria produced something useful he cannot say what in just four weeks.
The company is built around machine-learning programs that suggest changes to the genome which could produce an organism and setting—temperature, nutrient balance, and so on—that improves on the status quo. In this fiercely empirical process Zymergen makes DNA tweaks of all sorts, most of them to sequences that regulate gene expression. Arzeda, based in the Interbay district of Seattle, has a similar business model and is based on similar technologies.
Ginkgo, the i GEM -born startup in Boston, is another variation on the business-to-business theme. Its focus is not on the specifics of genome-based machine-learning or protein design, though it does both, so much as on developing a broader expertise in the remaking of microbes.
The three companies may differ in details of their approaches, but the big picture unites them. All of them see their current business-to-business approach as a stepping stone, a way of honing their techniques, teaching their machine-learning programs and bringing in cash as they develop products of their own. Arzeda talks of making tulipin, which among other things can greatly improve the qualities of perspex. Ginkgo is spinning out joint ventures with clients to work in specific areas. It has another spin-out working on cannabis, and has just announced a third one developing plant proteins for use in vegetarian foods, including meat substitutes.
Zymergen is looking at materials for electronics. They are also united in their zeal for high-throughput experiments. Their use of massive amounts of synthesised DNA is producing a new way of doing biology on an industrial scale. Today Ginkgo orders synthetic DNA sequences at 50, times that rate, using them to change the genomes of thousands of organisms a day. In it bought a DNA -synthesis company, Gen9, bringing all its production capacity in-house. That has not sated its appetite. Often these DNA sequences are not even looked at by humans before they arrive by courier.