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Isolation and functional analysis of a Kluyveromyces lactis RAP1 homologue
G P Larson1, D Castanotto, J J Rossi
1Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Gene
|December 2, 1994
Summary
Researchers identified a RAP1-like factor in Kluyveromyces lactis, crucial for ribosomal gene regulation. Despite conserved DNA-binding domains, Kluyveromyces lactis RAP1 cannot functionally replace Saccharomyces cerevisiae RAP1 in mutants.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- Saccharomyces cerevisiae RAP1 (Sc RAP1) is an essential protein regulating diverse genetic loci by binding to the UASRPG consensus sequence.
- Ribosomal protein genes in Kluyveromyces lactis (Kl) possess functional RAP1-binding elements, indicating a potential RAP1-like factor.
Purpose of the Study:
- To investigate the presence and characteristics of a RAP1-like factor in Kluyveromyces lactis.
- To compare the structure and function of Kluyveromyces lactis RAP1 (Kl RAP1) with Saccharomyces cerevisiae RAP1 (Sc RAP1).
Main Methods:
- Isolation of the Kl RAP1-encoding gene via homology screening.
- Amino acid sequence analysis and comparison of Kl RAP1 and Sc RAP1.
- Functional complementation assays using Sc rap1ts mutants.
Main Results:
- Kl RAP1 gene isolated, encoding a smaller protein (666 aa) than Sc RAP1 (827 aa) with a truncated N-terminus.
- DNA-binding domains are highly conserved, while N-termini and inter-domain regions are divergent.
- Kl RAP1 cannot functionally complement Sc rap1ts mutants, despite conserved DNA-binding domains.
Conclusions:
- Kluyveromyces lactis possesses a RAP1-like protein involved in ribosomal gene regulation.
- Structural divergence, particularly in non-DNA-binding regions, limits Kl RAP1's functional complementation of Sc RAP1.
- Kl RAP1 may have an autoregulatory role, as suggested by potential UASRPG elements in its 5' flanking region.