Composition and properties of trypsin-cleaved elongation factor Tu

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Elongation factor Tu (EF-Tu) is crucial for bacterial protein synthesis.
• EF-Tu facilitates the binding of aminoacyl-tRNA to ribosomes during translation.
• Proteolytic cleavage can alter protein structure and function.

Purpose of the Study:

• To investigate the effects of trypsin digestion on the structure and function of Escherichia coli EF-Tu.
• To identify the specific sites of trypsin hydrolysis and analyze the resulting fragments.
• To determine how proteolytic modification impacts EF-Tu's ability to stimulate protein synthesis and bind to tRNA and GTP.

Main Methods:

• Tryptic digestion of native EF-Tu.
• Analysis of digestion products using Edman degradation.
• Polyacrylamide gel electrophoresis (PAGE) for fragment separation.
• Assays for polyphenylalanine synthesis stimulation.
• Measurement of aminoacyl-tRNA binding to 70-S ribosomes.
• Assessment of ternary complex formation with aminoacyl-tRNA and GTP.

Main Results:

• Trypsin initially hydrolyzes EF-Tu at arginine-44, arginine-58, and lysine-56.
• A peptide fragment (Ala-45 to Arg-58) is excised, but protein synthesis stimulation activity is retained.
• Further hydrolysis produces an (A+D) fragment complex.
• This complex stimulates polyphenylalanine synthesis and aminoacyl-tRNA binding to ribosomes.
• The (A+D) complex forms a less stable ternary complex with aminoacyl-tRNA and GTP compared to native EF-Tu.

Conclusions:

• The excised peptide is not essential for EF-Tu's core function in stimulating protein synthesis.
• Limited tryptic digestion yields a functional EF-Tu fragment, suggesting domain flexibility.
• Proteolytic modification impacts the high-affinity binding of EF-Tu to the aminoacyl-tRNA-GTP ternary complex.