Mutations of the Mutations of the alternate start signal alternate start signal for the Galls protein in for the Galls protein in Agrobacterium rhizogenes Agrobacterium rhizogenes Henry McNett Henry McNett Dr. Walt Ream Dr. Walt Ream Department of Microbiology Department of Microbiology
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Mutations of the alternate start signal for the Galls protein in Agrobacterium rhizogenes Henry McNett Dr. Walt Ream Department of Microbiology.
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Mutations of the alternate start Mutations of the alternate start signal for the Galls protein in signal for the Galls protein in Agrobacterium rhizogenesAgrobacterium rhizogenes
Henry McNettHenry McNett
Dr. Walt ReamDr. Walt ReamDepartment of MicrobiologyDepartment of Microbiology
Importance of Agrobacterium Importance of Agrobacterium
Destroys thousands of plants all over the Destroys thousands of plants all over the world each yearworld each year
Is the only bacterium to our knowledge Is the only bacterium to our knowledge that actually inserts DNA into a eukaryotic that actually inserts DNA into a eukaryotic organismorganism
Provides very efficient way of transferring Provides very efficient way of transferring DNA into plant cellsDNA into plant cells
VirE2 and Single-Stranded T-DNA Are Exported VirE2 and Single-Stranded T-DNA Are Exported SeparatelySeparately
Galls replaces VirE2 proteinGalls replaces VirE2 protein
Agrobacterium rhizogenesAgrobacterium rhizogenes GALLS protein GALLS protein
Substitutes for Substitutes for A. tumefaciensA. tumefaciens single-stranded single-stranded DNA-Binding protein VirE2DNA-Binding protein VirE2
A. rhizogenes A. rhizogenes pRi1724pRi1724 encodes a protein encodes a protein that substitutes for VirE2that substitutes for VirE2
Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS
Galls differs from VirE2 Galls differs from VirE2
Galls contains an ATP binding motifGalls contains an ATP binding motif A helicase motifA helicase motif Has three repeats just after the nuclear Has three repeats just after the nuclear
localization signallocalization signal VirE2 is 533 AA whereas Galls is 1769 AAVirE2 is 533 AA whereas Galls is 1769 AA Both Galls and VirE2 have type 4 Both Galls and VirE2 have type 4
secretion signals secretion signals
Genetic representation of the Galls protein
GALLS-CT
•The c terminal part of the protein goes from The c terminal part of the protein goes from just after the nuclear localization signal to the c just after the nuclear localization signal to the c terminal end of the protein.terminal end of the protein.•We know this because mutations in the We know this because mutations in the nuclear localization signal do not affect the size nuclear localization signal do not affect the size of the c terminal fragment.of the c terminal fragment.•How is this other fragment being made?How is this other fragment being made?
Western blot of Galls proteinWestern blot of Galls protein
Western blot shows presence of shorter “C Western blot shows presence of shorter “C Terminus” fragment of the protein being made. Terminus” fragment of the protein being made.
Two possible ways C terminal Two possible ways C terminal fragment being madefragment being made
1) Alternate internal translation start site1) Alternate internal translation start site Translation could be starting internally as well as at the original start Translation could be starting internally as well as at the original start
codoncodon
2) Cleaving of the full length protein2) Cleaving of the full length protein The full length protein could be getting cleaved by the Agrobacterium to The full length protein could be getting cleaved by the Agrobacterium to
serve another function serve another function
Hypothesis:Hypothesis: The c terminal fragment is made by an internal translational start site.The c terminal fragment is made by an internal translational start site.
Project goalsProject goals
Test the hypothesis that the internal Test the hypothesis that the internal translational start site in the Galls protein translational start site in the Galls protein is used and is responsible for production is used and is responsible for production of the small c-terminal fragment.of the small c-terminal fragment.
BackgroundBackground
Alternate initiation site has 5 out of six base pairs required for an ideal Alternate initiation site has 5 out of six base pairs required for an ideal ribosome binding site. ribosome binding site.
Has methionine as start codon which is required for starting of translation of a Has methionine as start codon which is required for starting of translation of a proteinprotein
Ideal distance between start codon and ribosome binding site 7-9 base pairs, Ideal distance between start codon and ribosome binding site 7-9 base pairs, here there are 8.here there are 8.
The sequence for wild type Galls protein
RBS Start
Asp Ser Gly Glu Lys Asn Met Ala Ser 5'-GAC TCA GGA GAA AAA AAT ATG GCT TCG -3'
The mutation to the start codon
Methionine start codon
Methionine replaced by Isoleucine
Ribosome Binding Site
RBS Asp Ser Gly Glu Lys Asn Ile Ala Ser 5'-GAC TCA GGA GAA AAA AAT ATT GCT TCG -3'
Ribosome Binding Site
MethodsMethods
Alter coding region of Galls to change the Alter coding region of Galls to change the methionine coding to isoleucine methionine coding to isoleucine
Transform into Vir E2-deficient Transform into Vir E2-deficient Agrobacterium tumefaciensAgrobacterium tumefaciens
Use a western blot to detect the size(s) of Use a western blot to detect the size(s) of Galls being produced by Galls being produced by Agrobacterium Agrobacterium tumefacienstumefaciens
Schematic of Methods
Wild Type Galls plasmid cut with EcoR1 and Pst1
EcoR1 Pst1
Ligated in PCR product cut with EcoR1 and Pst1 with Methionine to Isoleucine mutation made in antisense primer
EcoR1
Pst1
Induced Cre facilitated recombination with broad host range vector for Agrobacterium tumefaciens Agrobacterium tumefaciens transformationtransformation
Transformation into
Agrobacterium tumefaciensAgrobacterium tumefaciens for protein analysis
Lox site
Lox site
EcoR1 Pst1
Met
Ile
Pst IPst IEcoRI
Pst IStep 1. Insert EcoRI fragment containing5‘ end of gene.
Step 2. Insert NcoIfragment containing3’ end of gene.
Step 3. Use Cre:lox site-specific recombination to insert plasmid containing rebuilt mutant GALLS gene into a plasmid capable of replicating in Agrobacterium tumefaciens.
ResultsResults
Ligated the Ligated the EcoEcoR1 fragment into plasmid R1 fragment into plasmid with correct orientationwith correct orientation
Ligated the Ligated the NcoNco1 fragment into plasmid 1 fragment into plasmid with correct orientationwith correct orientation
Cre:lox recombination of the plasmid Cre:lox recombination of the plasmid containing mutated containing mutated Galls Galls gene with broad gene with broad host range vectorhost range vector
Transform into VirE2 mutant strain of Transform into VirE2 mutant strain of Agrobacterium tumefaciensAgrobacterium tumefaciens
SummarySummary
Galls protein replaces function of VirE2 protein Galls protein replaces function of VirE2 protein into into Agrobacterium tumefaciensAgrobacterium tumefaciens
Galls protein is produced in two sizes: full length Galls protein is produced in two sizes: full length and a C-terminal fragment that may result from and a C-terminal fragment that may result from an internal translational start.an internal translational start.
Plasmid containing mutation of the putative Plasmid containing mutation of the putative internal translational start site is built and ready internal translational start site is built and ready for testingfor testing
Virulence assays and western blots are in Virulence assays and western blots are in progress.progress.
Special thanks Special thanks
Dr. Walt ReamDr. Walt Ream Howard Hughes Medical Institute Howard Hughes Medical Institute Larry Hodges Larry Hodges Dr. Kevin AhernDr. Kevin Ahern Deborah MoyerDeborah Moyer