The Effect of the 5’UTR
on in vivo Translation in
TYMV
Lisa Bauer
Microbiology
Mentor: Dr. Theo Dreher
Turnip Yellow Mosaic Virus (TYMV)

Genus: Tymovirus
 Single-stranded
positive-sense RNA
virus
 Found in dicotyledonous plants
 Viral Replication in all plant tissues

-Chloroplast membranes site of RNA
synthesis
 Why
study TYMV?
Genome structure of TYMV
DraI XmnI
p69
p206
5’UTR
 6318nt
cap
 3 overlapping reading frames (ORFs):
 -p69: Overlapping Protein
tRNA-like
Structure
CP
 5’ m7GppGMP


-p206: Replication Protein
-Coat Protein (CP)

Sub genomic expression
3’ UTR
5’UTR
*~…GCAAAUGAGUAAUGGCCU…
p69
p206
Objective: Determine how the structure of the 5’UTR of TYMV
affects which reading frame is most efficiently expressed in in
vivo translation.
Previous Studies:
Kozak, 1986:
-leaky scanning
Dreher, 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.
3’ tRNA-like structure
A
C
CUCU
U
A
C
G
U G
U
CCCG
CCC
GGGC
GGG
U
A
A
U
UCGGA AC C A
G
AGCCU
3´
-Val
eIF4E
UCA
A
C
G-C
AU U
A
U
C-G
A
C
G-C
A-U
U-A
A-U
C-G
GAUU G-C UCUUGAAU C
G-C
G
U-A
C-G
U-A

Major valine
G-C
U-A
identity nts in
C-G
C
the anticodon
C
A
C
loop
C A C
U
A
Enhanced translation with 3’UTR
seen in vivo (Matsuda et al., 2002)
Barends, 2003:
-TLS activates translation of
downstream reading frame p206
8 clones:
Experimental Constructs
Controls:
WT
p69
WT
p206
Generic 5’UTR (Luciferase)
WT 5’UTR
Pleij
p69
Pleij
p206
Shortened 5’UTR,
Pleij sequence
-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.
1. Linearize plasmid
LUC
2. In vitro run-off transcription
by T7 RNA polymerase
DM
(with cap analog)
3. RNA transfection
Daiki Matsuda
Protoplasts of
cowpea leaves
4. Cell lysis
DM
5. Luciferase reaction
5’UTR & 3’UTR Effect on Translational
-results corrected for LUC
Efficiency
specific activity
Jul 24
Generic 5’UTR
Ratio
1:
Generic/BamHI
1:
0.08
0.08
WT p69
1
2.70
WT p69/BamHI
WT p206
1:
0.20
0.08
WT p206/BamHI
Pleij p69
1.02
1:
Pleij p69/BamHI
0.16
Pleij p206
0.45
1:
Pleij p206/BamHI
0.08
0
2
4
With 3’UTR
6
Light Units
Ratio WT p69: Pleij p69 ~ 2.6:1
8
(x104)
10
No 3’UTR
Ratio WT p206: Pleij p206 ~ 0.44:1
5’UTR & 3’UTR Effect on Translational
Efficiency
Jul 24
Jul 30
Ratio
Generic 5’UTR
1:
1
1:
1:
0.08
Generic/BamHI
Ratio
1
0.09
0.08
1:
3.88
WT p69
0.10
4.97
WT p69/BamHI
WT p206
0.49
1:
WT p206/BamHI
0.08
0.17
Pleij p69
1.47
1:
Pleij p69/BamHI
0.10
1.09
1:
Pleij p206/BamHI
2
4
0.76
1:
0.08
0
2.20
1:
0.16
Pleij p206
0.96
1:
0.23
6
8
10
Light Units (x104)
0
2
4
6
Light Units (x104)
8
10
With 3’UTR
No 3’UTR
Ratio WT p69: Pleij p69 ~ 2.5:1
Ratio WT p206: Pleij p206 ~ 0.9:1
The Effect of Non-valylated 3’ ends
Aug 4
Ratio
1
Generic WT sg
0.107
Generic G56 sg
WT p69 sg
1
WT p69 G56 sg
0.126
WT p206 sg
1
WT p206 G56 sg
0.129
0
1
2
Light Units (x105)
-results corrected for LUC specific activity
3
4
WT 3’UTR
3’UTR without Val
The Effect of Non-valylated 3’ ends
Aug 4
Ratio
1
0.107
0.133
1
1
0.126
0.148
1
1
0.129
0.151
WT p69 sg
WT p69 G56 sg
Ratio
1
Generic WT sg
Generic G56 sg
Aug 8
WT p206 sg
WT p206 G56 sg
0
1
2
Light Units (x105)
3
4
WT 3’UTR
3’UTR without Val
0
1
2
3
5
Light Units (x10 )
4
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 Results
5’UTR constructs with and
without 3’TLS:
Ratio
1
Generic
Generic/DraI
Generic/BamHI
3.66
WT p69
WT p69/DraI
0.79
WT p206
WT p206/DraI
1.73
Pleij p69
Pleij p69/DraI
Pleij p206
1.06
Pleij p206/DraI
H2O
With TLS
0
2
4
6
8
10
12
Light Units (x105)
14
16
No TLS
No 3’UTR

Check luciferase activity in vitro of pLUC11
compared to constructs with 14 TYMV
amino acids