Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Ribonuclease T1 (RNase T1) is an enzyme crucial for RNA processing.
• Understanding the structure-function relationship of RNase T1 is essential for enzyme engineering and biotechnology.

Purpose of the Study:

• To chemically synthesize RNase T1 and its mutants.
• To investigate the functional importance of specific amino acid residues in RNase T1 activity.
• To elucidate the role of the wild-type triad and the guanosine recognition region.

Main Methods:

• Chemical synthesis of RNase T1 and human growth hormone genes.
• Expression of fusion proteins in Escherichia coli.
• Cleavage of fusion proteins using cyanogen bromide.
• Enzymatic activity assays to assess hydrolysis of pGpC bonds.

Main Results:

• A synthetic RNase T1 with a corrected Gly-Ser-Pro triad (residues 71-73) exhibited full enzymatic activity.
• A mutant with the erroneous Pro-Gly-Ser triad was inactive.
• Substitutions in the guanosine recognition region (residues 42-45) showed varied effects; Tyr to Phe substitutions had minor impact.
• Replacing Asn-43 with Arg or Ala caused minor activity reduction (>50% wild-type).
• Replacing Asn-44 with Asp or Ala drastically reduced activity to a few percent of wild-type.

Conclusions:

• The specific amino acid sequence Gly-Ser-Pro at residues 71-73 is critical for RNase T1 catalytic activity.
• Residue Asn-44 in the guanosine recognition region is vital for RNase T1 function.
• Chemical synthesis and site-directed mutagenesis are effective tools for studying enzyme structure-function relationships.