Joyce: Project overview: Difference between revisions
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===Background=== | ===Background=== | ||
tRNA nucleotidyltransferase catalyzes the transfer of CCA to the 3' end of a tRNA molecule, allowing the subsequent step of aminoacylation by an aminoacyl-tRNA synthetase. tRNA nucleotidyltransferase is therefore an essential gene | tRNA nucleotidyltransferase catalyzes the transfer of CCA to the 3' end of a tRNA molecule, allowing the subsequent step of aminoacylation by an aminoacyl-tRNA synthetase. tRNA nucleotidyltransferase is therefore an essential gene because it is required for aminoacylation and so translation. | ||
In ''Saccharomyces cerevisiae'', tRNA nucleotidyltransferase is targeted to the cytoplasm and the mitochondria. This targeting is achieved by the presence of three in-frame start codons. Proteins generated from ATG1 produce variants that go to the mitochondrion, while proteins generated from ATG2 or ATG3 are targeted to the cytosol. Removal of the first 9 or 17 amino acids didn't affect cytosolic targeting, but mitochondrial activity was reduced or lost | In ''Saccharomyces cerevisiae'', tRNA nucleotidyltransferase is targeted to the cytoplasm and the mitochondria. This targeting is achieved by the presence of three in-frame start codons (at amino acid positions 1, 10 and 18) which are initiated by three separate transcription start sites. Proteins generated from ATG1 produce variants that go to the mitochondrion, while proteins generated from ATG2 or ATG3 are targeted to the cytosol (and to a lesser degree, the nucleus). Removal of the first 9 or 17 amino acids didn't affect cytosolic targeting, but mitochondrial activity was reduced or lost; this implies that the first 9-17 amino acids play a significant role in mitrochondrial targeting. | ||
In ''Kluyveromyces lactis'', a yeast that is closely related evolutionary to ''S. cerevisiae'', tRNA nucleotidyltransferase is also targeted to the mitochondrion and cytosol. ''K. lactis'' tRNA nucleotidyltransferase was isolated by generating a temperature-sensitive ''CCA1'' mutant in ''S. cerevisiae'' and screening for temperature-resistant mutants by complementation with a ''K. lactis'' library. The identified sequence, ''KlCCA1'', showed a high level of identity with ''S. cerevisiae CCA1''. | NOTE TO SELF: ???This was determined by complementing a mutant of ''CCA1'' with the ORF of either variant and testing for growth on glucose (tests for cytoplasmic tRNA nucleotidyltransferase activity) or glycerol (tests for mitochondrial activity)???. This is possible because a cell that is growing on glucose must be using glycolysis to survive, and since glycolysis is purely cytoplasmic, it must be able to generate the proteins needed for glycolysis in the cytoplasm, which means it must have been able to translated effectively in the cytoplasm with a fully functional tRNA nucleotidyltransferase. For glycerol, the cell needs to undergo oxidative phosphorylation in the mitochondrion, and if the cell can grow, it must have a functional tRNA nucleotidyltransferase that is present in the mitocondrion. /NOTE | ||
In ''Kluyveromyces lactis'', a yeast that is closely related evolutionary to ''S. cerevisiae'', tRNA nucleotidyltransferase is also targeted to the mitochondrion and cytosol. ''K. lactis'' tRNA nucleotidyltransferase was isolated by generating a temperature-sensitive ''CCA1'' mutant in ''S. cerevisiae'' and screening for temperature-resistant mutants by complementation with a ''K. lactis'' library. The identified sequence, ''KlCCA1'', showed a high level of identity with ''S. cerevisiae CCA1'' and supported growth on both glucose and glycerol, suggesting that this protein was capable of mitochondrial and cytosolic transport. ''K. lactis'' has two in-frame start codons (at amino acid positions 1 and 4) which are | |||
CGATTCTTGCTTATGCTAAAATGTAGACTTGCTCTATCTTGAACAATCTAACACGTCATATATGGAATAC | |||
TTGTCAGCCAGTATGATCTTTATCGTCCACAATTGTGTCTTGCTTCCACTTTGGGTGGTATTTTGGAGCA | |||
GTTATCTGGTTAGCAGCAATTAAACAGATCTTATAATCTGACTGTTGAGCCTGATAGCGCGTCTAGTTGG | |||
CTTTCAGATCTATGTTTCATTGTATATACTCAGTCTTGACACATCTTATTTCAAGCCGTGGCCGATTTGA | |||
TACCAAGTTTTGTCCGATTAACGAGAATTTGTCCGGAGAAGTCGTTAAAATTGGTACGTGATTATCTCTG | |||
TGAAAACCCAACATTTCTATTTTGTTTCTTATCTGTTTCTACTTTTCATTCTGTTTCCACAACCTGATTT | |||
GTGTAAAAAACATCGGGTCACTTTCGAAGGAAAGGCTGAGATGTTATCACGTGATTTTGAATGTTACAAA | |||
TGGAACGCAATATCATGGTGACAGAATGACACCATCATGAATATACGGATATATATACTGAGGTTGCCAA | |||
TCGAATAAACCAGCACAACCCACTTCTGTTAGAGGATTTGCAAAGGAGCTAGTTACATACAGATAATGAG | |||
GATCCTTCATTGCTTAAGAACAGAACAAATCACGTGATTTGTAGTATATTAGCAGAACCACTTATATGTA | |||
CACACGTACACATATAGACACATATACCTTCAGTACAGCTGTTGTCGATGGTGCTCCCTAAGTAGGTGTT | |||
TCTGAGCGCAAATTGGCTTGGAAGCTGACGTTTCTAGTTCGGACTCACTACCGTATTCCCGCATTACCCT | |||
ACCGTCTATATGCCTAATCTTTCCTATTTCAACATGTACTCATCATCTCATCTCATCTCACTTCATCACA | |||
TCAAATCACATCACATCACAGCTTATCTTCTGTCCAAGGCAACCACTCCACTCTCTAGAGCATATTGTAT | |||
CTCAACAATTATACTCGACC'''ATG'''TTCAAA'''ATG'''GTAGCGTCGAAGATACAGTTGAATAAAGTTGAATCGGA | |||
AATATGTACGCTGGTGAAAGAGTTCTGTTCTCATTATAATAAAGCGAATGCAGAGACTGAACCTTTGGTA | |||
GCCCGAATCACCGGAGGTTGGGTCCGTGACAAGTTGTTGGGAAATGATTCTAAT'''GATCTAGAT'''ATTGCAA | |||
TTAATAAC'''ATG'''ACGGGTGAGCAATTCGCTGAAAAACTATGTGCTTTCCTGCAAGATCGGGGGTTAGAAAC | |||
GCATTCTTTACATACCATTGATAAAAACCCATCGAAATCTAAACATTTGGAGACTTGTACCACGAAACTC | |||
TTCGACGTTCCAGTGGATTTCGTTAATTTGAGATCAGAAGAGTACACAATGGAATCTAGGATCCCGAAAG | |||
TGGAATTTGGAACCCCATACGATGATGCGATGCGACGTGATGCCACTTTGAACGCTATGTTCTACAATAT | |||
AACGGAGGATAAAATCGAAGACTTTACTAAGAAAGGGTTTCAGGATTTGAACGATGGAATATTACGTACT | |||
CCTTTACCACCAAGACAAACTTTCATAGATGATCCTTTACGTGTTCTGAGATTGATTAGATTTGCATCAA | |||
GATTCAATTTCCAAATTGACCCGCAGACTTATCAAGCAATGAGGGATCCTGGTATCCACCAGTCCTTCAA | |||
CCATAAGATTTCCAAAGGTCGTGTATATACCGAAATGCACAAAACTCTTACATCTGCTAATCCCTTTTAC | |||
GCATTGGATTTAATTCAAGGTGCTCATCTTTCCAGAGTTATTTTCACCACTAATGAAAGTTCTCCTGAAA | |||
TCGAATCCATTTATGAGAACCTGGACCAGCATTTAAAGAGTTTAGTAGAAACAATACCAAAACTTTTAAA | |||
ATCACACACAACATTTGCTAGTGTTTTCCCGGGCATGCAGGAGCCTTTGATATTATCACTTGTATTATCT | |||
GGATTCAAAGGATTAAAGGGCCCAGATCCAGCAAAGCCTAAAAATTCGATACCTTTAGCTGGCGTTATTA | |||
CAAAAGAAGGCTTAAACTTCCCTAATACTCAAGTAGACAACGTTATCGCTTGTGTTGAATCTGAGGATTC | |||
CTATCATAATTTAGTTAAGAATGGAAAATCAATGAAAAGATCGGAGTTGGGTTTTGCTTTGAGAAAACTT | |||
GGTAAGAATTGGCAAATGGTTCATTTCTATAATCTATGTTTGGATTATTTACGCCATGGAGATGAGCCAA | |||
TTCCACATTATGACGAATTTTATAAGCATGTCCACGATTGCAAACTAGACGATGTCTATACTTTAAAGCA | |||
TATCATCAACGGTAAAGAACTAGCGAAATTACTGGATAGGAAACCTGGCATTTGGATGGGAGAAACATTA | |||
GACAGAATCCTTATTTGGCAACTAGATAATCCAGATATTTCGAAAGAAACCTTTATTGAAAACCTTAATG | |||
ACATAGTACATCTTCCTTGA |
Revision as of 09:56, 12 September 2009
Joyce May-December 2009
Mapping the transcription start sites of Kluyveromyces lactis tRNA nucleotidyltransferase
Background
tRNA nucleotidyltransferase catalyzes the transfer of CCA to the 3' end of a tRNA molecule, allowing the subsequent step of aminoacylation by an aminoacyl-tRNA synthetase. tRNA nucleotidyltransferase is therefore an essential gene because it is required for aminoacylation and so translation.
In Saccharomyces cerevisiae, tRNA nucleotidyltransferase is targeted to the cytoplasm and the mitochondria. This targeting is achieved by the presence of three in-frame start codons (at amino acid positions 1, 10 and 18) which are initiated by three separate transcription start sites. Proteins generated from ATG1 produce variants that go to the mitochondrion, while proteins generated from ATG2 or ATG3 are targeted to the cytosol (and to a lesser degree, the nucleus). Removal of the first 9 or 17 amino acids didn't affect cytosolic targeting, but mitochondrial activity was reduced or lost; this implies that the first 9-17 amino acids play a significant role in mitrochondrial targeting.
NOTE TO SELF: ???This was determined by complementing a mutant of CCA1 with the ORF of either variant and testing for growth on glucose (tests for cytoplasmic tRNA nucleotidyltransferase activity) or glycerol (tests for mitochondrial activity)???. This is possible because a cell that is growing on glucose must be using glycolysis to survive, and since glycolysis is purely cytoplasmic, it must be able to generate the proteins needed for glycolysis in the cytoplasm, which means it must have been able to translated effectively in the cytoplasm with a fully functional tRNA nucleotidyltransferase. For glycerol, the cell needs to undergo oxidative phosphorylation in the mitochondrion, and if the cell can grow, it must have a functional tRNA nucleotidyltransferase that is present in the mitocondrion. /NOTE
In Kluyveromyces lactis, a yeast that is closely related evolutionary to S. cerevisiae, tRNA nucleotidyltransferase is also targeted to the mitochondrion and cytosol. K. lactis tRNA nucleotidyltransferase was isolated by generating a temperature-sensitive CCA1 mutant in S. cerevisiae and screening for temperature-resistant mutants by complementation with a K. lactis library. The identified sequence, KlCCA1, showed a high level of identity with S. cerevisiae CCA1 and supported growth on both glucose and glycerol, suggesting that this protein was capable of mitochondrial and cytosolic transport. K. lactis has two in-frame start codons (at amino acid positions 1 and 4) which are
CGATTCTTGCTTATGCTAAAATGTAGACTTGCTCTATCTTGAACAATCTAACACGTCATATATGGAATAC
TTGTCAGCCAGTATGATCTTTATCGTCCACAATTGTGTCTTGCTTCCACTTTGGGTGGTATTTTGGAGCA
GTTATCTGGTTAGCAGCAATTAAACAGATCTTATAATCTGACTGTTGAGCCTGATAGCGCGTCTAGTTGG
CTTTCAGATCTATGTTTCATTGTATATACTCAGTCTTGACACATCTTATTTCAAGCCGTGGCCGATTTGA
TACCAAGTTTTGTCCGATTAACGAGAATTTGTCCGGAGAAGTCGTTAAAATTGGTACGTGATTATCTCTG
TGAAAACCCAACATTTCTATTTTGTTTCTTATCTGTTTCTACTTTTCATTCTGTTTCCACAACCTGATTT
GTGTAAAAAACATCGGGTCACTTTCGAAGGAAAGGCTGAGATGTTATCACGTGATTTTGAATGTTACAAA
TGGAACGCAATATCATGGTGACAGAATGACACCATCATGAATATACGGATATATATACTGAGGTTGCCAA
TCGAATAAACCAGCACAACCCACTTCTGTTAGAGGATTTGCAAAGGAGCTAGTTACATACAGATAATGAG
GATCCTTCATTGCTTAAGAACAGAACAAATCACGTGATTTGTAGTATATTAGCAGAACCACTTATATGTA
CACACGTACACATATAGACACATATACCTTCAGTACAGCTGTTGTCGATGGTGCTCCCTAAGTAGGTGTT
TCTGAGCGCAAATTGGCTTGGAAGCTGACGTTTCTAGTTCGGACTCACTACCGTATTCCCGCATTACCCT
ACCGTCTATATGCCTAATCTTTCCTATTTCAACATGTACTCATCATCTCATCTCATCTCACTTCATCACA
TCAAATCACATCACATCACAGCTTATCTTCTGTCCAAGGCAACCACTCCACTCTCTAGAGCATATTGTAT
CTCAACAATTATACTCGACCATGTTCAAAATGGTAGCGTCGAAGATACAGTTGAATAAAGTTGAATCGGA
AATATGTACGCTGGTGAAAGAGTTCTGTTCTCATTATAATAAAGCGAATGCAGAGACTGAACCTTTGGTA
GCCCGAATCACCGGAGGTTGGGTCCGTGACAAGTTGTTGGGAAATGATTCTAATGATCTAGATATTGCAA
TTAATAACATGACGGGTGAGCAATTCGCTGAAAAACTATGTGCTTTCCTGCAAGATCGGGGGTTAGAAAC
GCATTCTTTACATACCATTGATAAAAACCCATCGAAATCTAAACATTTGGAGACTTGTACCACGAAACTC
TTCGACGTTCCAGTGGATTTCGTTAATTTGAGATCAGAAGAGTACACAATGGAATCTAGGATCCCGAAAG
TGGAATTTGGAACCCCATACGATGATGCGATGCGACGTGATGCCACTTTGAACGCTATGTTCTACAATAT
AACGGAGGATAAAATCGAAGACTTTACTAAGAAAGGGTTTCAGGATTTGAACGATGGAATATTACGTACT
CCTTTACCACCAAGACAAACTTTCATAGATGATCCTTTACGTGTTCTGAGATTGATTAGATTTGCATCAA
GATTCAATTTCCAAATTGACCCGCAGACTTATCAAGCAATGAGGGATCCTGGTATCCACCAGTCCTTCAA
CCATAAGATTTCCAAAGGTCGTGTATATACCGAAATGCACAAAACTCTTACATCTGCTAATCCCTTTTAC
GCATTGGATTTAATTCAAGGTGCTCATCTTTCCAGAGTTATTTTCACCACTAATGAAAGTTCTCCTGAAA
TCGAATCCATTTATGAGAACCTGGACCAGCATTTAAAGAGTTTAGTAGAAACAATACCAAAACTTTTAAA
ATCACACACAACATTTGCTAGTGTTTTCCCGGGCATGCAGGAGCCTTTGATATTATCACTTGTATTATCT
GGATTCAAAGGATTAAAGGGCCCAGATCCAGCAAAGCCTAAAAATTCGATACCTTTAGCTGGCGTTATTA
CAAAAGAAGGCTTAAACTTCCCTAATACTCAAGTAGACAACGTTATCGCTTGTGTTGAATCTGAGGATTC
CTATCATAATTTAGTTAAGAATGGAAAATCAATGAAAAGATCGGAGTTGGGTTTTGCTTTGAGAAAACTT
GGTAAGAATTGGCAAATGGTTCATTTCTATAATCTATGTTTGGATTATTTACGCCATGGAGATGAGCCAA
TTCCACATTATGACGAATTTTATAAGCATGTCCACGATTGCAAACTAGACGATGTCTATACTTTAAAGCA
TATCATCAACGGTAAAGAACTAGCGAAATTACTGGATAGGAAACCTGGCATTTGGATGGGAGAAACATTA
GACAGAATCCTTATTTGGCAACTAGATAATCCAGATATTTCGAAAGAAACCTTTATTGAAAACCTTAATG
ACATAGTACATCTTCCTTGA