1. The Effect of the 5’UTR on in vivo Translation in TYMV Lisa Bauer Microbiology Mentor: Dr. Theo Dreher

2. 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?

3. p69 CP 3’ UTR Genome structure of TYMV tRNA-like Structure p206 5’UTR  6318nt  5’ m7GppGMP cap  3 overlapping reading frames (ORFs): - p69 : Overlapping Protein -p206: Replication Protein -Coat Protein (CP)  Sub genomic expression XmnIDraI

4. 5’UTR 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. Objective: Determine how the structure of the 5’UTR of TYMV affects which reading frame is most efficiently expressed in in vivo translation. *~…GCAAAUGAGUAAUGGCCU… p69p206

5. 3’ tRNA-like structure U A A U G-C C-G G-C A-U G-C G U-A C-G U-A G-C U-A C-G A C U A C C C A A C U C G U CCCG GGGC CCC GGG CUCU UCGGAA AGCCU UCA UG 3´ GAUU C-G U-A G-C U U U A A A C A-U UCUUGAAU C CCAC l Major valine identity nts in the anticodon loop G -Val Enhanced translation with 3’UTR seen in vivo (Matsuda et al., 2002) eIF4E AA A C CC C Barends, 2003: -TLS activates translation of downstream reading frame p206

6. Experimental Constructs 8 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

7. Experimental Process Polymerase chain reaction (PCR) Digestion with Not I and Pst I enzymes Ligation Transformation  E.coli Purify DNA & Sequence

8. 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 Daiki Matsuda

9. 5’UTR & 3’UTR Effect on Translational Efficiency Light Units (x10 4 ) 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: 0.08 1: 0.08 1: 0.08 1: 0.08 1: 0.16

10. 5’UTR & 3’UTR Effect on Translational Efficiency Ratio WT p69: Pleij p69 ~ 2.5:1Ratio 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: 0.08 1: 0.08 1: 0.08 1: 0.08 1: 0.16 Light Units (x10 4 ) 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 24Jul 30 With 3’UTR No 3’UTR

11. Light Units (x10 5 ) 0 1 2 3 4 Aug 4 The Effect of Non-valylated 3’ ends WT 3’UTR 3’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

12. 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’UTR 3’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 (x10 5 ) 0 1 2 3 4 Aug 4Aug 8

13. Conclusion The 1 st 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.

14. 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

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

16. Preliminary Results 5’UTR constructs with and without 3’TLS: Light Units (x10 5 ) 0 2 4 6 810 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

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