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

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

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

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

  4. 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.

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

  6. Experimental Constructs 8 clones: Controls: WT p69 WT p206 Generic 5’UTR (Luciferase) WT 5’UTR Pleij p69 Shortened 5’UTR, Pleij sequence Pleij p206 -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. 1. Linearize plasmid LUC 2. In vitro run-off transcription by T7 RNA polymerase (with cap analog) DM 3. RNA transfection Daiki Matsuda Protoplasts of cowpea leaves 4. Cell lysis DM 5. Luciferase reaction

  9. 5’UTR & 3’UTR Effect on Translational Efficiency -results corrected for LUC specific activity Jul 24 Ratio 1 2.70 0.20 1.02 0.45 Generic 5’UTR 1: 0.08 1: 0.08 Generic/BamHI WT p69 WT p69/BamHI WT p206 1: 0.08 WT p206/BamHI Pleij p69 1: 0.16 Pleij p69/BamHI Pleij p206 1: 0.08 Pleij p206/BamHI With 3’UTR 0 2 4 6 8 10 No 3’UTR Light Units (x104) RatioWT p69: Pleij p69~ 2.6:1 RatioWT p206: Pleij p206~ 0.44:1

  10. 5’UTR & 3’UTR Effect on Translational Efficiency Jul 24 Jul 30 Ratio 1 3.88 0.49 1.47 1.09 Ratio 1 4.97 0.96 2.20 0.76 Generic 5’UTR 1: 0.09 1: 0.08 1: 0.08 Generic/BamHI 1: 0.10 WT p69 WT p69/BamHI WT p206 1: 0.08 1: 0.17 WT p206/BamHI Pleij p69 1: 0.16 1: 0.10 Pleij p69/BamHI Pleij p206 1: 0.08 1: 0.23 Pleij p206/BamHI 0 2 4 6 8 10 0 2 4 6 8 10 Light Units (x104) Light Units (x104) With 3’UTR No 3’UTR RatioWT p69: Pleij p69~ 2.5:1 RatioWT p206: Pleij p206~ 0.9:1

  11. The Effect of Non-valylated 3’ ends Aug 4 Ratio 1 0.107 Generic WT sg Generic G56 sg WT p69 sg 1 0.126 1 0.129 WT p69 G56 sg WT p206 sg WT p206 G56 sg 0 1 2 3 4 Light Units (x105) WT 3’UTR -results corrected for LUC specific activity 3’UTR without Val

  12. The Effect of Non-valylated 3’ ends Aug 4 Aug 8 Ratio 1 0.133 1 0.148 1 0.151 Ratio 1 0.107 1 0.126 1 0.129 Generic WT sg Generic G56 sg WT p69 sg WT p69 G56 sg WT p206 sg WT p206 G56 sg 0 1 2 3 4 0 1 2 3 4 Light Units (x105) Light Units (x105) WT 3’UTR 3’UTR without Val

  13. 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.

  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: Ratio 1 3.66 0.79 1.73 1.06 Generic Generic/DraI Generic/BamHI WT p69 WT p69/DraI WT p206 WT p206/DraI Pleij p69 Pleij p69/DraI Pleij p206 Pleij p206/DraI H2O With TLS 0 2 4 6 8 10 12 14 16 No TLS Light Units (x105) No 3’UTR

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

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