Spinach chloroplast RNase P: a putative protein enzyme

Area of Science:

• Molecular Biology
• Enzymology
• Plant Biochemistry

Background:

• Ribonuclease P (RNase P) is crucial for tRNA maturation, typically involving an RNA subunit in bacteria and eukaryotes.
• Previous studies suggested spinach chloroplast RNase P might differ due to its properties inconsistent with RNA presence.

Purpose of the Study:

• To biochemically characterize highly purified Ribonuclease P (RNase P) from spinach chloroplasts.
• To determine if chloroplast RNase P activity resides in RNA or protein and its catalytic mechanism.

Main Methods:

• Extensive purification of spinach chloroplast RNase P (500-1500 fold).
• Gel filtration chromatography (Sephacryl S-200 and S-300) to determine enzyme mass.
• Crosslinking studies with iodoUMP-substituted pre-tRNA to identify the active subunit.
• Assays using Rp-thio-substituted pre-tRNA to investigate metal ion requirements.

Main Results:

• Purified chloroplast RNase P exhibits enzymatic activity associated with a ~70 kDa polypeptide.
• Gel filtration indicates an approximate mass of 70 ± 5 kDa for the active enzyme.
• The enzyme efficiently cleaves pre-tRNA without requiring divalent metal ions like Mg2+ or Mn2+.

Conclusions:

• Spinach chloroplast RNase P is a protein-based enzyme, functioning as a conventional protein enzyme rather than a ribozyme.
• This finding contrasts with the well-established ribozyme nature of RNase P in bacteria and other eukaryotic organelles.