Parallel dimerization of a PrrC-anticodon nuclease region implicated in tRNALys recognition

Area of Science:

• Bacteriology
• Molecular Biology
• Biochemistry

Background:

• Escherichia coli restriction tRNase PrrC is a bacterial antiviral enzyme.
• PrrC has a functional C-domain and a regulatory N-domain.
• The C-domain's role in tRNA recognition is not fully understood.

Purpose of the Study:

• To investigate the involvement of a specific C-domain sequence in tRNA(Lys) recognition.
• To understand the structural basis of PrrC's tRNA binding and cleavage activity.

Main Methods:

• Synthesized a peptide mimic (LARP) of the C-domain sequence.
• Used UV-cross-linking to assess LARP's interaction with tRNA(Lys) anticodon stem-loop (ASL) analogs.
• Investigated the effect of LARP modifications and PrrC mutations on activity.
• Performed cysteine-tethering experiments and site-directed mutagenesis to study dimerization.

Main Results:

• LARP mimicked the C-domain's ability to bind tRNA(Lys) ASL and inhibit PrrC cleavage.
• Peptide trimming or inactivating mutations reduced LARP's activity.
• Cysteine tethering to LARP enhanced its activity, suggesting parallel dimerization.
• Mutations in the C-domain corresponding to LARP induced intersubunit cross-links, confirming parallel C-domain dimerization.

Conclusions:

• A specific peptide sequence within PrrC's C-domain is crucial for tRNA(Lys) recognition.
• PrrC's C-domains dimerize in parallel, contributing to tRNA binding.
• The enzyme's structure suggests distinct assembly stages for its NTPase and tRNA-binding sites.