Phage

genome replication by R oppen up A RBS site and make A translation available, but only available during the initial stage of + strand synthesis.

Phage

• MS2
  • small +ssRNA
    • 3569nts
    • 4 ORFs with intergenic spaces
      • A
        • maturation protein that recognize sex pilis
      • CP
        • major coat protein
      • L
        • Lysis protein
        • overlapping with CP and R, with out itself's RBS
      • R
        • Replicase
    • high mutation rate
    • RNA secondary structures regulates RBS of A, CP, R
      • CP
        • first only CP RBS is available
        • increases exponentially during the replication cycle.
      • R
        • CP translation scanning oppen up secondary structure, makes R's RBS available
        • made in increasing amounts in first 20 minutes.
      • L
        • L is some time translated when robosome scan through it start codon
        • made in small amount
      • A
        • genome replication by R oppen up A RBS site and make A translation available, but only available during the initial stage of + strand synthesis
        • only made later and in small amounts.
  • host range
    • Gram- with sex(F) pilis
  • Capsid
    • CP
    • A
• M13
  • linear +ssDNA
    • Circular supercoiled ~6400nt ssDNA genome
    • encode 11 proteins
      • 5 capsid proteins
        • major coat protein
          • pVIII
            • (+) charged carboxy-termini of pVIII bind (–) charged phosphate groupson viral genomic DNA
        • minor coat proteins on 2 ends of the helix
          • adsorption tip to recognize bacterial F pilus
            • pIII
            • PVI
          • base that initiates assembly
            • pVII
            • pIX
      • 3 DNA synthesis proteins
        • II, V, X
      • 3 assembly proteins
        • I, iV, XI
    • 3 IGs
      • major
        • replication ori
        • packaging sequence
          • Hari pin
          • efficient phage assmbly
        • rho-dependent transcriptional terminator
          • a frequently transcribed region from gene II to VIII
      • minor
        • strong transcriptional terminator
          • an infrequently transcribed region from gene III to IV
    • replication
      • rolling cycle
  • host range
    • E coli
  • capsid
    • 900nm long & 9nm in diameter helical structure: no head
  • different penetration and release methods
    • penetration
      • entire viron enter, but not only DNA
    • release
      • phages extrude from bacteria membrane but not lysis
  • application
    • • Cloning vectors for genetic manipulation, DNA sequencing and Site-directed mutagenesis. • Phage display - Engineering Gene III or Gene VIII to produce new peptides.
• λ
  • 48.5 kb linear dsDNA
    • cII control lytic/ lysogenic cycle
      • it self's transcription and translation are controlled by N anti-terminator protein
      • In metabolically inactive cell, cII protein is stable and activates transcription of cI. CI protein, acting as an repressor, can silence the entire λ genome except the genes for integration. The lysogenic cycle maintains
      • In metabolically active cell, cII protein is not stable, could not activate the expression of cI, so that the late genes (starting from Q) can be expressed. phage enters into lytic cycle
  • Capsid
    • Icosahedral head
    • long, flexible, non-contractile tail with a central tail fiber and side tail fibers
  • application
    • Cloning Vectors (in vitro DNA packaging) · Phage vectors - insertion or replacement. · Cosmid vectors (Plasmids bearing λ cos sites).
• adsorption and penetration
  • host surface "receptors"
    • Phage T4
      • adsorption via host surface lipopolysaccharides
        • require Na+
    • phage lamda
      • adsorption via E. coli maltose receptor protein LamB
    • phage chi
      • adsorption via flagella
        • require Mg2+
    • Phage M13
      • adsorption via E coli sex F pili tip
    • Phage MS2
      • adsorption via gram- sex F pili
  • procedure
    • 1. long tail fibre adsorp host surface
    • 2. Tail pin adsorp host surface
    • 3. Tail sheats contracts and release needle with in it
    • 4. the needle penetrates plasma membrane and injects viral genome
      • 3' end first
• tricky df
  • IG region
    • Non-coding region, or untranslated region