The small subunit (?2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2(III)-Y(?)) that initiates nucleotide reduction in the large subunit (a2) via a long range radical transfer (RT) pathway in the holo-(a2)m(?2)n complex. The C-terminal tails of ?2 are predominantly responsible for interaction with a2, with a conserved tyrosine residue in the tail (Tyr(356) in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the ? tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (??'), of which only ? contains a cofactor, to address both of these issues. We demonstrate that neither ?-Tyr(376) nor ?'-Tyr(323) (Tyr(356) equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-??' and an in vivo viability assay show that ?-Tyr(376) is essential for RT, whereas Tyr(323) in ?' is not. Although the C-terminal tail of ?' is dispensable for cofactor formation and RT, it is essential for interactions with ? and a to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the ? and ?' C-terminal tails relative to a within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the ? ?' interface.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|