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