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Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain
X Zhao1, B Georgieva, A Chabes
1Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, New York, New York 10032, USA.
Abstract:
In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.
Insights
Lethality in budding yeast mutants mec1 and rad53 is suppressed by mutations in the SML1 gene. These mutations abolish Sml1 protein interaction with ribonucleotide reductase, indicating this interaction causes lethality.
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- MEC1 and RAD53 are essential for cell growth in budding yeast.
- Lethality of mec1 or rad53 mutants is suppressed by the absence of Sml1, a ribonucleotide reductase (RNR) inhibitor.
- The Sml1-Rnr1 interaction's role in this suppression requires further investigation.
Purpose of the Study:
- To investigate the relationship between Sml1-Rnr1 interaction and suppression of mec1/rad53 lethality.
- To identify specific SML1 mutations that alleviate mec1/rad53 inviability.
- To elucidate the structural and functional significance of the Sml1-Rnr1 interaction.
Main Methods:
- Random mutagenesis of the SML1 open reading frame.
- Analysis of SML1 mutant protein expression levels.
- Assessment of mec1 and rad53 strain viability.
- Mapping of mutations within the SML1 protein.
- Nuclear magnetic resonance (NMR) studies to reveal protein structure.
- Deletions within SML1 to assess RNR inhibitory activity.
- Interaction studies with yeast Rnr3 and human R1.
Main Results:
- Seven viable SML1 mutations were identified in mec1 and rad53 strains without affecting protein expression.
- All seven mutations abolished the interaction between Sml1 and Rnr1.
- The identified mutations cluster in the C-terminal 33 amino acids of Sml1, with four residues in an alpha-helical region.
- N-terminal deletions of Sml1 did not affect RNR inhibition.
- Mutations disrupted interactions with both yeast Rnr3 and human R1.
Conclusions:
- The Sml1-Rnr1 interaction is directly responsible for the lethality observed in mec1 and rad53 budding yeast strains.
- The C-terminal region of Sml1, particularly an alpha-helix, is crucial for Rnr1 binding.
- The binding mechanism between Sml1 and the RNR large subunit is conserved across species.