Download RNA Phage

Viroids
- very small (246-375 nucleotides) ssRNA
- highly structured (resistant to ribonuclease)
- do not code for any proteins
- do not require helper virus
- infected tissue contains no virus-like particles
- replicated by host cell machinery (but RNA!!!)
When compare strains and natural recombinants:
- find conserved functional domains
PSTVd potato spindle tuber viroid
Theodor O. Diener
RNA Phage - MS2
Small genome - 3,569 nucleotides (+ve ssRNA)
Genome encodes single coat protein + 3 others
Icosahedral (26nm)
For each progeny virion, replication needs to produce :
1
genome
180 x coat protein
1 x
maturation protein (A)
Replicase must be made early
Lysis protein must be made late
Need to prevent collisions between replicase and ribosomes
Production of 20,000 progeny viruses / infected cell in 22 mins
Most of genome base paired in 2o structure (resistant to ribonuclease)
T3
Single coat protein
1.
Grow E. coli cells to a
concentration ~ 2.0x109
cfu/ml (O.D. ~ 2-3); very
turbid
2.
Infect bacterial culture
with low concentration
(1000 pfu/ml) of RNA
phage - MS2
3.
Record O.D. in 20 minute
time intervals
Yellow = control (uninfected
bacteria show exponential
growth)
Red = bacteria infected with
RNA phage – MS2 virus
Organization of genes
5'
A maturation
COAT
REPLICASE
LYSIS
Availability of RBS
Availability of RBS
1) Small amount of coat protein translated
- opens 2o structure in genome
- allows replicase translation (but not lysis protein)
Replicase complexes with 3 host proteins
2) Replicase binds to 5' end of coat protein gene
- no ribosomes on +ve strand to block replicase
3) Make -ve and +ve copies of genome
- translates one A protein each time +ve strand initiated.
- dilutes replicase
- then make coat protein from new +ve strands
4) Coat protein
- acts as repressor of replicase gene
- binds +ve genome strand, 2o changes - lysis protein translated
- assembly of progeny
A protein is required for phage attachment to male E. coli pilus + entry of RNA
For MS2, protein levels are controlled by Initiation of TRANSLATION
(availability of RBS)