The Effect of the 5and 3UTRs on in vivo translation in TYMV.pptVIP

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 6318nt 5’ m7GppGMP cap 3 overlapping reading frames ORFs : -p69: Overlapping Protein -p206: Replication Protein -Coat Protein CP Sub genomic expression 5’UTR 3’ tRNA-like structure Experimental Constructs Experimental Process Polymerase chain reaction PCR Digestion with Not I and Pst I enzymes Ligation Transformation E.coli Purify DNA Sequence Experimental Process Cont. 5’UTR 3’UTR Effect on Translational Efficiency The Effect of Non-valylated 3’ ends 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 Check luciferase activity in vitro of pLUC11 compared to constructs with 14 TYMV amino acids * * p69 CP 3’ UTR tRNA-like Structure p206 5’UTR XmnI DraI 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. *~…GCAAA