Bacteriophage DNA Packaging RNA Gears in a DNA Transport Machine

main actor in this play is the “portal” or “head-tail conThe question of how the dsDNA bacteriophages get nector,” which is an annular structure made of 12 copies their genomic DNA on the inside of the virus particle, (possibly 13 copies in some cases) of the phage-encoded surrounded by their protein capsid on the outside, has portal protein (Carazo et al., 1986; Dube et al., 1993). been onthe minds of virologists probably since Salvador The portal resembles a grommet, sitting on one of the Luria and Thomas Anderson made the first electron mi5-fold symmetric “corners” of the icosahedral capsid. crographs of phage T2 and saw tadpole-shaped objects There is a roughly DNA-sized hole in the middle of the that they likened to spermatozoa (Luria and Anderson, portal, and in the mature phage this hole is lined up with 1942). Initially it seemed obvious that the DNA must first the hole down the middle of the tail. When the DNA is condense into a compact form, after which the capsid injected into a new host, it must pass through the hole proteins would come together to form a shell around it. in the portal on its way down the tail and into the cell. The problem was framed in its current form about 25 For DNA packaging (which happens before the tail is on years ago when it was shown—astonishingly at the the scene) most models envision the DNA entering the time—that during the latter stages of a phage infection, capsid through the hole in the portal as well, though an empty protein shell is assembled first, and then the there is neither universal agreement nor direct evidence DNA is somehow transported across the shell into the on this point. The portal is thought to be the place where interior (Luftig et al., 1971; Kaiser et al., 1975). Studies the terminase binds during packaging, and the termiaimed at understanding this processhave had important nase–portal complex, with the DNA passing through it, technological benefits for the field—think of phage l is generally considered to be the heart of the machine cosmid vectors and in vitro DNA packaging—but the that moves the DNA. The terminase is an ATPase and DNA packaging process also has a number of features therefore most likely the first molecule in the chain of that recommend it as a model system for teasing out energy transduction that leads to DNA transport. A rethe secrets of some of the most central problems of markable ability of the DNA packaging machine is to modern molecular biology. These include how protein– sense how much DNA has been packaged into the head protein interactions contribute to biological function, and “decide” when the head is full and it is time to trigger how protein–DNA interactions produce movement in a the terminal cleavage of the DNA. Genetic experiments multicomponent structure, and more generally, how a implicate the portal in this process (Casjens et al., 1992; complex biological machine is able to carry out an intriTavares et al., 1992). cate task: what are all the molecular gears and levers, One of the technical advances that has made it possiand how do they work together to carry out their role—in ble to learn much of what we know about DNA packaging this case to pump the DNA up a steep thermodynamic is the development of in vitro packaging systems. Such gradient into the capsid? As described below, there systems have been worked out for a number of phages, are now clear indications that RNA has a specific and and as they have become better defined and more effiessential role in this process as well. cient it has been possible to show, for example, that The phage biologists who have addressed these isDNA packaging is driven by ATP hydrolysis to the tune sues over the years have worked out a rather detailed of about one ATP per two base pairs translocated into picture of the overall process of DNA packaging (see, the capsid (Guo et al., 1987b). One of the best such in for example, Black, 1988). Briefly, the product of phage vitro packaging systems is that of the Bacillus subtilis DNA replication, which may be a multigenome concaphage φ29, and it was with this system that the startling temer or a single-genome-sized molecule depending on discovery was reported several years ago that there is the phage, interacts with a phage-encoded protein to an absolute requirement for presence of an RNA moleinitiate the packaging process. In the case of a typical cule in order to see packaging (Guo et al., 1987a). The concatemer-forming phage like the well-studied l, the RNA in question, termed “packaging RNA” or “pRNA,” protein in question is the heterodimeric enzyme termiis a 174-nucleotide molecule encoded in the phage genase, which binds site-specifically to the DNA and cuts it nome. A highly folded secondary structure initially posto form the first mature end of the soon-to-be-packaged tulated from sequence gazing has been largely supDNA. The terminase stays with the DNA after cutting, ported by mutational analysis and by phylogenetic and the terminase–DNA complex finds its way to a proanalysis of the apparently homologous pRNA sequences head—the emptyprotein shell that has formed in a sepafrom a number of phages closely related to φ29. The rate assembly pathway—andbinds. Insertion of the DNA pRNA is found associated with the prohead in about six into the prohead commences, cut end first, and contincopies, but it is completely absent from the mature viues until the head is full of DNA. During DNA insertion rion, and in that sense can be considered an essential