Date of Award
1993
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
The zero length crosslink between the inhibitory {dollar}\epsilon{dollar} subunit and one catalytic {dollar}\beta{dollar} subunit of E. coli F{dollar}\sb1{dollar}-ATPase ({dollar}\alpha\sb3\beta\sb3\gamma\delta\epsilon{dollar}), induced by 1-ethyl-3- ((3-dimethylamino)propyl) -carbodiimide (EDC), was determined. A crosslinked CNBr fragment derived from {dollar}\beta{dollar}-{dollar}\epsilon{dollar} was identified by SDS-PAGE. Protein sequence analysis revealed the constituent peptides to be {dollar}\beta{dollar}-Asp-380--Met-431 and {dollar}\epsilon{dollar}-Glu-96--Met-138. {dollar}\beta{dollar}-Glu-381 was absent from cycle 2 indicating that it was one of the crosslinked residues, but no potential crosslinked residue in {dollar}\epsilon{dollar} was identified.;An {dollar}\epsilon{dollar} mutant with methionine replacing Val-112 ({dollar}\epsilon{dollar}V112M) was produced, incorporated into F{dollar}\sb1{dollar}, then crosslinked with EDC. A crosslinked CNBr fragment of {dollar}\beta{dollar}-{dollar}\epsilon{dollar}V112M was shown to contain the peptides {dollar}\epsilon{dollar}-Glu-96--Met-112 and {dollar}\beta{dollar}-Asp-380--Met-431. Again {dollar}\beta{dollar}-Glu-381 was notably reduced and no missing residue from the {dollar}\epsilon{dollar} peptide could be identified, but the peptide sequence limited the prospects to Ser-106, Ser-107 or Ser-108. Another {dollar}\epsilon{dollar} mutant with cysteine replacing Ser-108 (S108C), could not be crosslinked to {dollar}\beta{dollar} in F{dollar}\sb1{dollar} by EDC. These results indicated that the {dollar}\beta{dollar}-{dollar}\epsilon{dollar} crosslink in F{dollar}\sb1{dollar} occurred between {dollar}\beta{dollar}-Glu-381 and, likely, {dollar}\epsilon{dollar}-Ser-108. As these residues must be located immediately adjacent to one another in F{dollar}\sb1{dollar}, these results define a contact site between {dollar}\beta{dollar} and {dollar}\epsilon{dollar}.;The mechanism of translational control of expression of the uncC gene ({dollar}\epsilon{dollar} subunit) relative to the immediately 5{dollar}\sp\prime{dollar} uncD gene ({dollar}\beta{dollar} subunit) was determined. In F{dollar}\sb1{dollar} {dollar}\beta{dollar}:{dollar}\epsilon{dollar} stoichiometry is 3:1, but the polycistronic unc mRNA contains single copies of each gene, implying translational control of expression. Early expression studies and a computer analysis suggested an RNA secondary structure including the 3{dollar}\sp\prime{dollar} end of uncD and the uncDC intergenic region. Analysis of synthetic mRNA by cleavage with RNases and by chemical labelling confirmed the predicted structure. Premature uncD stop codons inserted within the secondary structure showed little effect on {dollar}\epsilon{dollar} expression, whereas stop codons situated 5{dollar}\sp\prime{dollar} of the structure showed significant reductions, indicating that translational control of {dollar}\epsilon{dollar} synthesis involved partial coupling to {dollar}\beta{dollar} synthesis via disruption of the stem-loop by ribosomes translating uncD. The relatively small size of the structure ({dollar}\sim{dollar}100 bases) may ensure rapid re-folding compared to translation initiation at uncC, and keep {dollar}\epsilon{dollar} synthesis low compared to {dollar}\beta{dollar}.
Recommended Citation
Dallmann, Helmut Garry, "Structural And Biosynthetic Relationships Between The Beta And Epsilon Subunits Of Escherichia Coli F(1)-atpase" (1993). Digitized Theses. 2294.
https://ir.lib.uwo.ca/digitizedtheses/2294