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Bacteriophage M13

Bacteriophage M13 is a filamentous (as opposed to icosahedral) virus that infects F+ E. coli.

Bacteriophage M13 infects F+ E. coli, but rather than killing host cells it just slows growth. Virions leak out from the cell. Thus, the virus is not very lytic.

M13 has a 6kb circular ssDNA genome.. Special proteins at the tips are involved in assembly, morphogenesis, adsorption and penetration.

M13 absorbs the tip of the F-pilus and injects its genome into the cytoplasm of the infected cell.

Immediately after injecting the viral DNA into the cytoplasm, the circular ssDNA is converted into dsDNA. The dsDNA is transcribed by host RNAP into viral mRNAs. These mRNAs are translated by host cell ribosomes into viral proteins.

Proteins then direct replication of viral DNA by host cell enzymes. Progeny virons assemble.

Converting ssDNA to dsDNA

  • Strand synthesis following infection was blocked by treateing cells with E. coli RNAP inhibitor.
  • Do you need pre-existing RNAP for ssDNA → dsDNA? Find out if new viral gene expression is not needed for M13 genome replication.
    • Infect cells in presence of chloramphenicol (inhibits new protein synthesis).
    • Result: dsDNA is still made.
    • Conclusion: No new protein synthesis required for synthesis of viral dsDNA.
    • Transform naked viral ssDNA into cells.
    • Result: a small amount is converted to dsDNA.
    • Conclusion: capsid proteins not involved.
These studies revealed that the single stranded viral DNA becomes coated with E. coli Ssb (single-stranded DNA binding protein), except in one region where the sequence forms a hairpin structure.

E. coli RNA polymerase can initiate RNA synthesis at this position on the M13 + strand because it is sufficiently double-stranded for RNA polymerase to use as a template. Once the - strand RNA primer is synthesized by RNA polymerase, it is extended around the circle of the + template strand by E. coli DNA polymerase III: Ssb is released as the - strand is synthesized. The RNA primer is then removed by digestion by the 5' to 3' exonuclease activity of DNA polymerase I. The primer is replaced by the DNA polymerase activity of DNA Pol I.

Finally, the DNA ends are ligated together by DNA Ligase.

Converting dsDNA back to ssDNA

We are trying to take the ssDNA, get a capsid around it, and then get it out of the cell.

We initially have circular dsDNA. pII knicks the + strand and binds to the end of the + strand. This allows DNAPIII and helicase together duplicate the strand to make another + strand. When pV accumulates late in infection, it binds to ssDNA and replaces the ssDNA binding protein. pVIII binds to the inner membrane. After pV coats the DNA, pVII and PIX binds to the morphogenic sequence. At the same time, pI and pIV compose a secretion apparatus in the membrane. pV interacts with pVIII and the DNA is passed through the secretion apparatus, with pVIII replacing pV. pIII and pVI are added to the tips of the DNA.

At the end, there is one strand of DNA with pII, pIII, PVI and pVIII bound to it.

Protein synthesis

  • pI comes a secretion apparatus, it is involved in making a pore in the membrane.
  • pII first knicks the dsDNA and binds to the end of the + strand. From that, DNAPolIII and helicase replicate the + strand.
  • pIII (along with pVI) forms the rounded tip, and is the first protein to interact with E. coli.
  • pIV (along with pI) form the secretion appartus, thus making a pore in the membrane. pI and pIV make a pore.
  • pV displaces ssDNA binding protein. It interacts with (and is dispaced by) pVIII, thus guiding the virion toward and through the secretion appartus.
  • pVI (along with pII) forms the rounded tip, and is one of the first proteins to interact with E. coli.
  • pVII (along with pIX) bind to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.
  • pVIII lines the inner membrane. It interacts with (and replaces) pV as the phage exits the cell. The secreted virion is surrounded by pVIII rather than pV.
  • pIX (along with pVII) binds to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.

Questions

How do you initiate DNA replication from a dsDNA circle? How do you separate ssDNA circles needed for further DNA replication from those to be packaged? How do you put a capsid around the DNA and get it out of the cell? How do you initiate transcription from the dsDNA circle?
  • Protein II (pII) nicks the + strand.
How do you separate ssDNA circles needed for further DNA replication from those that are to be packaged?
  • pV accumulated and binds displaced _ strand in place of El coli Ssb. = strand bound by pV does not serve as a template for - strand synthesis. M13 + strand replication...describe the cycle in detail.

At the origin of replication, a hairpin structure is formed that allows host cell RNAP to bind. RNAP lays down RNA primers. Possible ways for M13 to express genes: host cell zynes, virus encodes enczome packaged in capsid, encodes during lytic cycle

SSDNABindigProtein = SSB

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