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BIOL 4010 Study Guide - Final Guide: Myc, Antigenicity, Ribonuclease

Course Code
BIOL 4010
Andrew Lang
Study Guide

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Section 2- Bacteriophages
Differentiation of the three different tailed phage families
dsDNA Order:
- Long thick, contractile rigid tails
- Discovered in 1951 by Esther Lederberg
- Long, non-contractile, flexible tails
- Released from E.coli strain K12 where it is a prophage
- Entire gene sequence is coded in 1 mRNA and is organized in a
pattern much like its structure in the virion
- I 99, Hedi & Duda disoeed that lada PaPa ildtpe
was a recombinant version
- Ur- has tail fibres and is the original strain
- Discovered through genome sequencing
- Tail fibre genes in PaPa had a frameshift mutation
- Phage with tail fibres adsorbs to cells better and will bind and
infect cells with glucose
- Receptors on the cells are recognized by J proteins on the tail
(LamB) which is a maltose transporter, expressed in the presence
of maltose (higher infection with maltose)
- All particles have same tail length controlled by protein H
- Size of H protein is directly proportional to the size of the tail
- Genome has 12 nucleotide ssDNA ends called cos sites which
circularize when entering the cell
- After infection 2 genes are expressed N and Cro
- Expression of recombination and DNA replication genes
If Lysogeny: 2 genes are express, C1 & int
DNA integrates and C1 is only expessed
1. N and Cro proteins translated
2. Translation stops at terminator
3. N binds at nut sites and continues translation
4. Next bunch of genes are translated including CII
5. CII activates expression of CI and integrase
6. Integrase recombines genome into the host genome at the AttP
and attB sites
7. CI forms dimers and binds to Operator sites and stops gene
If no CI present then virus enters the lytic cycle
- Short, non-contractile tails
- Model lytic virus
- Icosahedral capsid, 3 core protiens, portal protein (opening), 2 tail
proteins, and 6 tail fibres
- Tail fibres bind to the LPS on outside of the cells (bops around the
surface of the cell until contact is made with multiple tail fibres)
- Core proteins pushed through tail & cell wall, only 850bp enter the
cell (begins to be transcribes before entire molecule is in)
- Genome pulled into cell by action of cellular RNA polymerase
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(transcribing class 1 genes)
- Class 1:
Inactivation of host RNA polymerase
Inactivation of host restriction enzyme
Makes virus RNA polymerase (very different)
- Transcription of class 2 genes by virus RNA polymerase pulls more
of the genome into the cell
- Class 2:
Degradation of host DNA
Replication of phage DNA
- Nested transcription start sites in class 2 genes creates
oeRNA’s fo late lass  gees
- Class 3 is last to enter cell and last to be transcribed but binds the
best to RNA polymerase (class 2 protein aids in binding)
- Class 3:
Virion structure and assembly
Structural proteins and lysis proteins
- DNA replication generates copy of DNA with small gaps at the end
that are recombined into a concatemer
- Cut up into single genomes during DNA packaging
- T7 is used as a tool for molecular biology
Move promoter sequence for T7 RNA polymerase infront
of a gene of interest
Turn on the polymerase, very active and generates
desired genes at high amounts
Ur-lambda versus lambda-PaPa and function of tail fibers
- A lot of work was done with PaPa which does not have tail fibres realized 1992
- PaPa is a recombinant version and was selected because of large plaque size
- Genome sequence in 1982 and tail fibre genes were present but not active because of a frame
shift mutation
- Hendrix and Duda in 1992 stated PaPa as ot the othe of all phages
- They resurrected the original from K12 and observed tail fibres
- Ur- adsorbs to the cell better than PaPa and it will bind and infect cells grown in glucose
- the presence of a J protein on the tail fibres allows recognition of the receptor LamB which is a
maltose transporter only expressed in the presence of maltose
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Different states of prophages for different phages, e.g. N15 and P1 versus lambda
- There are prophages (plasmid prophages) which do not integrate into the bacterial genome
- Needs two things: (P1)
- Partition genes (ensure that each daughter gene gets one copy of the plasmid)
- Plasmid addiction module (Doc and Phd)
o Doc- stable toxin, which induces cell death and is transferred into every daughter cell
o Phd is an unstable inhibitor of Doc , which degrades in daughter cells without plasmid
o If daughte ell loses plasid the Phd degades a’t e eplaed ad Do eoes
- Coliphage N15 DNA circularizes after entering the cell at cos sites, circular form the linearlizes
with a different form than in the virus particle
- Linear form does not integrate into the genome but develops telomeres (hairpin ends)
- Half of the genome is the same as lambda, the other half is very different and codes for genes
which allow the DNA to exist as a linear plasmid
- Some genes allow replication as a circular plasmid
Functions of S, R and Rz/Rz1 in lambda lysis
- Lysis genes are S, R and Rz/Rz1 act as molecular clocks to trigger lysis only at appropriate times
- S= holin (puts a whole in the cytoplasmic membrane)
o Has 2 forms: S107 (anti-holin) and S105 (holin)
o Extra 2 amino acids prevent helix 1 from entering the membrane, and inhibits holin
o Holin accumulates in the CM over time, reaches critical conc and membrane
depolarizes, conformation changes to form a hole, kills cell
- R= endolysin (digests the peptidoglycan)
o A glycosidase transglycosylase and breaks down sugar linkages
o Different phages use different enzymes
- Rz/Rz1 = spanin (disrupts the outer membrane)
o Overlapping genes for 2 proteins in different reading frames (o-spanin and i-spanin)
o Rz attaches to the inner membrane, RZ1 is a lipoprotein attached to the inner side of the
outer membrane
o The 2 interact and disrupt the outer membrane (by pinching together, makes hole)
Relationships of SAPIs and GTAs to phages
- SaPIs= staphylococcus aureus pathogenicity island
- Different SaPIs are present in different strains and encode for various virulence factors
- 15 kb long and normally not active but are induced for application by phages
- Produce proteins that hijack phage capsid proteins and packaging machinery
- Lysis results in release of some original helper phage and SaPIs with shrunken heads
- SaPIs encode a repressor protein (Stl) and helper phage proteins inhibit the repressor and
activate SaPIs
- Gene transfer agents that are encoded in the bacterial genome that resemble phage structures
- Seem to have evolved from prophages and likely arose independently in different organisms
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