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Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects
Summary
Key residues Lys-16 and Asp-119 are crucial for Ha-ras guanine nucleotide binding. Altering these sites impacts ras protein function, cellular transformation, and yeast cell viability, offering insights into ras protein regulation.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- The Ha-ras protein is a key regulator of cellular processes.
- Guanine nucleotide binding is essential for ras protein activity.
- Specific amino acid residues are implicated in modulating ras function.
Purpose of the Study:
- To investigate the roles of Lys-16 and Asp-119 in Ha-ras guanine nucleotide binding.
- To determine the functional consequences of mutations at these residues on cellular transformation.
- To propose a structural model for the GDP/GTP-binding site of Ha-ras.
Main Methods:
- Site-directed mutagenesis to alter Lys-16 and Asp-119 residues in Ha-ras.
- Measurement of guanine nucleotide binding affinity and specificity.
- Microinjection of purified proteins into NIH 3T3 cells to assess cellular transformation.
- Analysis of Ha-ras protein function in yeast cells.
Main Results:
- Lys-16 substitution (Asn) reduced Ha-ras affinity for GDP/GTP 100-fold, without altering specificity.
- Asp-119 substitution (Ala) reduced Ha-ras affinity for GDP/GTP 20-fold and specificity.
- [Ala119]Ha enhanced cellular transformation compared to wild-type Ha.
- Mutations affecting nucleotide binding showed dominant temperature-dependent lethality in yeast, mitigated by coexpression of RAS2.
Conclusions:
- Lys-16 and Asp-119 are critical for Ha-ras guanine nucleotide binding and biological function.
- Altered nucleotide binding affinity influences cellular transformation and yeast cell viability.
- A structural model for the Ha-ras GDP/GTP-binding site is proposed based on mutation data.