Isolation and functional analysis of a Kluyveromyces lactis RAP1 homologue.
Saccharomyces cerevisiae RAP1 (Sc RAP1) is an essential protein which interacts with diverse genetic loci within the cell. RAP1 binds site-specifically to the consensus sequence, 5'-AYCYRTRCAYYW (UASRPG, where R = A or G, W = A or T, Y = C or T). In Kluyveromyces lactis (Kl) ribosomal protein-encoding genes (rp) retain functional RAP1-binding elements, suggesting the presence of a RAP1-like factor. Kl extracts display an activity capable of specifically binding to rp fragments bearing UASRPG. We subsequently isolated the Kl RAP1-encoding gene by homology to a subfragment which encodes the N terminus of the DNA-binding domain of Sc RAP1. The predicted amino acid (aa) sequence of Kl RAP1 indicates it is smaller than Sc RAP1 (666 vs. 827 aa) with the N terminus being truncated. The DNA-binding domain is virtually identical between the two RAP1 proteins, while the RIF1 domain is moderately conserved. The region between these two domains and the N-termini are highly divergent. Two potential UASRPG were identified in the 5' flanking region, suggesting an autoregulatory role for RAP1. Despite the similarities between the two proteins, KI RAP1 is unable to complement Sc rap1ts mutants, suggesting that domains essential for function in Sc are absent from the Kl protein.
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
G P
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Larson
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Castanotto
J J
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Rossi
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Malafa
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020668:rap GTP-Binding Proteins
D005656:Fungal Proteins; D012269:Ribosomal Proteins; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins