Ty3 lifecycle maturation Nuclear pore translocation txn assembly Nucleus integration translation...
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Transcript of Ty3 lifecycle maturation Nuclear pore translocation txn assembly Nucleus integration translation...
Ty3 lifecycle
maturation
Nuclear pore translocation
txn
assembly
Nucleus
integration
translation
Gag3
Gag3-Pol3
export
uncoating
reversetranscription
cDNA
VLP
Pheromoneinduction
Ste12
GLFG Nups
TFIIIB, TFIIIC
Imt1-4Xrn1Dhh1Lsm1Eap1Pub1Stm1Tma20Hcr1
Ty3 expression systems
α
α factor inducible endogenous promoter
galactose inducible synthetic promoter
LTR GAG3 POL3GAL10 GAG3 POL3
Gag3 Mutagenesis
Larsen and Zhang
SP
wt
Dhh1-GFP
Ty3-RFP
Merge
Basic
K271AK272AE273A Dbasic DNC
Zinc
C267A C280AC267AC280A
* *NC (p7)
C267
A
C280
A
NC determines Gag3-Pol3 association with P bodies
IN-GFP
m7G AAAAA
Dhh1-GFP
Plasmid
cDNA
WT G207DH233V
G207D/H233V
WT PR-ΔAcidic
Δacidic PR-
ΔSP
CA SP NC
H233VG207D
Gag3
ATTHLQEEYDSDYDSEDNLTQNGYVH
ΔSP/ΔAcidic
•Blocking SP-NC blocks subsequent processing and cDNA synthesis (G207D)
•Blocking CA-SP blocks transposition after cDNA synthesis (H233V)
•Neutralization of the acidic residues of SP causes failure to condense the immature particle (Dacidic)
•DSP produces particles and cDNA, but fails to transpose
•Therefore particle structure requires a negative charge on SP, but the essential function is after cDNA synthesis, for example in uncoating
Localization with P body proteins does not insure packaging of GAG3-POL3 RNA
DDX6 Family of DExD/H RNA Helicases
—Implicated in splicing, ribosome biogenesis, RNA transport, degradation, and translation
— ATP binding and hydrolysis activities, RNA binding activity.— Facilitate unwinding of double stranded RNA; thought to act as a remodeler of mRNP
complexes
—Evolutionarily conserved p54 family: DDX6, p54, RCK (human), Xp54 (X. laevis), CGH-1 (C. elegans) dhh1 (S. cerevisae) and others
— promotes RNA decapping and translational repression in yeast— component of both stress granule and P body mRNP complexes in yeast— Component of germ granules in C. elegans; protects maternal mRNAs during oogenesis. — Dhh1 and RCK/hDhh1 are required for efficient translation o f positive strand RNA viruses BMV
and HCV, respectively.
**Recent data has demonstrated that Dhh1p has weaker RNA-dependent ATPase activity than other DEAD-box helicases; Dhh1p helicase activity was not observed in vitro.
Q I Ia Ib II III IV V VI
REC A-like Domain 1 Rec A-like Domain 2
dhh1Δ lsm1Δ stm1Δ xrn1Δ
uncoatingtranslocation,integrationDhh1
Xrn1Dhh1Xrn1 Xrn1
Lsm1Stm1
Proteins associated with Ty3 Gag3 affect retrotransposition intermediates