The Effect of the 5’UTR on in vivo Translation in

TYMV

Lisa BauerMicrobiology

Mentor: Dr. Theo Dreher

Turnip Yellow Mosaic Virus (TYMV)

Genus: TymovirusSingle-stranded positive-sense RNA

virusFound in dicotyledonous plantsViral Replication in all plant tissues

-Chloroplast membranes site of RNA synthesis

Why study TYMV?

p69

CP

3’ UTR

Genome structure of TYMV

tRNA-like Structure

p2065’UTR

6318nt 5’ m7GppGMP cap 3 overlapping reading frames (ORFs):

-p69: Overlapping Protein -p206: Replication Protein -Coat Protein (CP)

Sub genomic expression

XmnIDraI

5’UTR

Previous Studies: Kozak, 1986:

-leaky scanningDreher, 1989:

-in vitro translation showed proteins of two reading frames equally produced

Pleij et al., 1997:-the secondary structure of the 5’UTR does not affect the

expression of the two different ORFs in vitro

Hypothesis: The two different ORFs will be equally expressed in vivo.

Objective: Determine how the structure of the 5’UTR of TYMV affects which reading frame is most efficiently expressed in in vivo translation.

*~…GCAAAUGAGUAAUGGCCU…

p69 p206

3’ tRNA-like structure

UAAU

G-CC-GG-CA-UG-CGU-AC-GU-AG-CU-AC-G

AC

UA

CCC

AA

C U

C GU

CCCG

GGGC

CCC

GGG

CUCU

UCGGA A

AGCCU

UCAU G

3´

GAUUC-GU-AG-C

UUU

AAA

CA-U

UCUUGAAU C

CC AC

Major valine identity nts in the anticodon loop

G-Val

Enhanced translation with 3’UTR seen in vivo (Matsuda et al., 2002)

eIF4E

AA

AC

CC

C

Barends, 2003:

-TLS activates translation of downstream reading frame p206

Experimental Constructs8 clones:

Shortened 5’UTR, Pleij sequence

WT 5’UTR

WT p69

Pleij p69

WT p206

Pleij p206

Generic 5’UTR (Luciferase)

Controls:

-WT p69, WT p206 & Pleij p206 made by Katie Tinnesand

Experimental Process

Polymerase chain reaction (PCR) Digestion with Not I and Pst I enzymes Ligation Transformation

E.coli Purify DNA & Sequence

Experimental Process Cont.

2. In vitro run-off transcription by T7 RNA polymerase(with cap analog)

1. Linearize plasmid

3. RNA transfection

4. Cell lysis

5. Luciferase reaction

LUC

Protoplasts of cowpea leaves

DM

DM

Daiki Matsuda

5’UTR & 3’UTR Effect on Translational Efficiency

Light Units (x104)

Jul 24

0 2 4 6 8 10

Ratio

1

2.70

0.20

1.02

0.45

Ratio WT p206: Pleij p206 ~ 0.44:1Ratio WT p69: Pleij p69 ~ 2.6:1

WT p69

WT p69/BamHI

WT p206

WT p206/BamHI

Pleij p69

Pleij p69/BamHI

Pleij p206

Pleij p206/BamHI

Generic 5’UTR

Generic/BamHI

With 3’UTR

No 3’UTR

-results corrected for LUC specific activity

1:

0.081: 0.08

1:

0.08

1:

0.08

1:

0.16

5’UTR & 3’UTR Effect on Translational Efficiency

Ratio WT p69: Pleij p69 ~ 2.5:1 Ratio WT p206: Pleij p206 ~ 0.9:1

WT p69

WT p69/BamHI

WT p206

WT p206/BamHI

Pleij p69

Pleij p69/BamHI

Pleij p206

Pleij p206/BamHI

Generic 5’UTR

Generic/BamHI

Ratio

1

3.88

0.49

1.47

1.09

1:

0.081:

0.08

1:

0.08

1:

0.08

1:

0.16

Light Units (x104) Light Units (x104)

0 2 4 6 8 10 0 2 4 6 8 10

1:

0.09

1:

0.10

1:

0.10

1:

0.17

1:

0.23

Ratio

1

4.97

0.96

2.20

0.76

Jul 24 Jul 30

With 3’UTR

No 3’UTR

Light Units (x105)

0 1 2 3 4

Aug 4

The Effect of Non-valylated 3’ ends

WT 3’UTR3’UTR without Val

Generic WT sg

WT p69 sg

WT p69 G56 sg

WT p206 sg

WT p206 G56 sg

Generic G56 sg

Ratio

1

0.107

1

0.126

1

0.129

-results corrected for LUC specific activity

The Effect of Non-valylated 3’ ends

Generic WT sg

WT p69 sg

WT p69 G56 sg

WT p206 sg

WT p206 G56 sg

Generic G56 sg

WT 3’UTR3’UTR without Val

Ratio

1

0.107

1

0.126

1

0.129

Ratio

1

0.133

1

0.148

1

0.151

Light Units (x105) Light Units (x105)0 1 2 3 4

0 1 2 3 4

Aug 4 Aug 8

Conclusion The 1st reading frame is most efficiently

translated (WT p69 & Pleij p69) for both long and short 5’UTR

Translation of TYMV more efficient with presence of 3’TLS, except in WT p69

The 3’ effect on translational efficiency is independent of the 5’ effect.

In Progress

Re-construct Pleij p69 & Pleij p206 to match our version of TYMV 5’UTR sequence used in WT p69 & WT p206

Knock out the alternate AUG

Acknowledgements

Dr. Dreher Daiki Matsuda Katie Tinnesand Kevin Ahern Howard Hughes Medical Institute

Preliminary Results5’UTR constructs with and without 3’TLS:

Light Units (x105)

0 2 4 6 8 10 12 14 16

WT p69

WT p69/DraI

WT p206

WT p206/DraI

Pleij p69

Pleij p69/DraI

Pleij p206

Pleij p206/DraI

H2O

Generic

Generic/DraI

Generic/BamHI

With TLS

No TLS

Ratio

1

3.66

0.79

1.73

1.06

No 3’UTR

Check luciferase activity in vitro of pLUC11 compared to constructs with 14 TYMV amino acids