M13 Particle... The major coat protein is the product of phage gene 8 (g8p) and there are 2,700 - 3,000 copies of this protein per particle, together with approximately 5 copies each of four minor capsid proteins, g3p, g6p, g7p and g9p which are located at the ends of the filamentous particle. The primary structure of the major coat protein g8p explains many of the properties of the particle. Mature molecules of g8p consist of approximately 50 amino acid residues (a signal sequence of 23 amino acids is cleaved from the precursor protein during its translocation into the outer membrane of the host bacterium), and is almost entirely alpha-helical in structure so that the molecule forms a short rod. There are three distinct domains within this rod: A negatively-charged region at the amino terminal end which contains acidic amino acid residues and which forms the outer, hydrophilic surface of the virus particle A basic, positively-charged region at the carboxyl terminal end which lines the inside of the protein cylinder adjacent to the negatively-charged DNA genome A hydrophobic region which is responsible for interactions between the g8p subunits which allow the formation of and stabilize the phage particle. Ff phage particles are held together by the hydrophobic interactions between the coat protein subunits and this is demonstrated by the fact that the particles fall apart in the presence of chloroform, even though they do not contain any lipid component. The g8p subunits in successive turns of the helix interlock with the subunits in the turn below, are tilted at an angle of approximately 20° to the long axis of the particle and have been described as overlapping one another like the scales of a fish. The value of µ (protein subunits per complete helix turn) is 4.5 and p (axial rise per subunit) = 1.5nm. Since the phage DNA is packaged inside the core of the helical particle, the length of the particle is dependent on the length of the genome. In all Ff phage preparations the following forms occur: Polyphage: containing more than one genome length of DNA Miniphage: deleted forms containing 0.2-0.5 phage genome lengths of DNA Maxiphage: genetically defective forms but containing more than one phage genome length of DNA This plastic property of these filamentous particles has been exploited by molecular biologists to develop the M13 genome as a cloning vector - insertion of foreign DNA into the non-essential inter-genic region results in recombinant phage particles which are longer than the wild-type filaments. Unlike most viruses, there is no sharp cut-off genome-length at which the genome can no longer be packaged into the particle. However, as M13 genome size increases, the efficiency of replication declines such that while recombinant phage genomes 1-10% longer than the wild-type do not appear to be significantly disadvantaged, those 10-50% longer than the wild-type replicate significantly more slowly and above 50% increase over the normal genome length it becomes progressively more difficult to isolate recombinant phage. This property has been exploited in M13 cloning vectors
Posted on: Wed, 26 Mar 2014 12:47:40 +0000
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