Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease

Area of Science:

• Molecular Biology
• Enzymology
• RNA Biochemistry

Background:

• Colicin E5 from Escherichia coli is a ribonuclease with a specific cleavage activity.
• This activity is localized to its C-terminal domain (CRD).
• The enzyme targets specific transfer RNAs (tRNAs) by cleaving their anticodon loops.

Purpose of the Study:

• To elucidate the recognition mechanism of the Colicin E5 C-terminal domain (E5-CRD).
• To identify the specific sequence determinants for E5-CRD cleavage.
• To understand the substrate specificity of Colicin E5.

Main Methods:

• Utilized synthetic minihelix RNAs mimicking tRNA anticodon loops.
• Assessed the susceptibility of these minihelices to E5-CRD.
• Determined the minimal substrate required for E5-CRD specificity.

Main Results:

• E5-CRD cleaves tRNAs (tRNA(Tyr), tRNA(His), tRNA(Asn), tRNA(Asp)) at the anticodon loop, recognizing a UQU sequence.
• The specificity determinant for E5-CRD is the YGUN motif (-1 to +3 relative to the anticodon).
• The minimal substrate retaining specificity for E5-CRD is GpUp, with loop structure influencing cleavage efficiency.

Conclusions:

• The YGU motif is essential for E5-CRD recognition and cleavage.
• The identity of the nucleotide at position N (in YGUN) dictates the degree of minihelix susceptibility.
• Structural constraints imposed by contiguous nucleotides are overcome by specific loop formations in substrate recognition.