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, 14 (8), 1558-67

Footprinting Analysis Demonstrates Extensive Similarity Between Eukaryotic RNase P and RNase MRP Holoenzymes

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Footprinting Analysis Demonstrates Extensive Similarity Between Eukaryotic RNase P and RNase MRP Holoenzymes

Olga Esakova et al. RNA.

Abstract

Eukaryotic ribonuclease (RNase) P and RNase MRP are evolutionary related RNA-based enzymes involved in metabolism of various RNA molecules, including tRNA and rRNA. In contrast to the closely related eubacterial RNase P, which is comprised of an RNA component and a single small protein, these enzymes contain multiple protein components. Here we report the results of footprinting studies performed on purified Saccharomyces cerevisiae RNase MRP and RNase P holoenzymes. The results identify regions of the RNA components affected by the protein moiety, suggest a role of the proteins in stabilization of the RNA fold, and point to substantial similarities between the two evolutionary related RNA-based enzymes.

Figures

FIGURE 1.
FIGURE 1.
Examples of the footprinting assays. (A) Fe-EDTA footprinting of RNase MRP. (Lane 1) deproteinated RNase MRP RNA control (no Fe-EDTA); (lane 2) holoenzyme control (no Fe-EDTA); (lane 3) deproteinated RNase MRP RNA (Fe-EDTA); (lane 4) holoenzyme (Fe-EDTA); (lanes 5,6) sequence marker (sequenced RNase MRP RNA). Numbers on the right show nucleotide numbering. Primer RTP4 was used in the primer extension reactions. (B) DMS footprinting of RNase MRP. (Lane 1) deproteinated RNase MRP RNA control (no DMS); (lane 2) holoenzyme control (no DMS); (lanes 3–5) deproteinated RNase MRP RNA (various concentrations of DMS); (lanes 6–8) RNase MRP holoenzyme (various concentrations of DMS); (lanes 9,10) sequence marker (sequenced plasmid DNA). Numbers on the right show nucleotide numbering. Primer RTP25 was used in the primer extension reactions.
FIGURE 2.
FIGURE 2.
Regions of RNase MRP (A) and RNase P (B) RNAs affected by protein binding in the holoenzyme. Red markings signify higher level of protection in the holoenzyme versus deproteinated RNA; green markings indicate no change in sensitivity upon deproteination; blue markings indicate lower sensitivity upon deproteination. Highlighting of nucleotides describes the results of the Fe-EDTA footprinting assays (indicative of the exposure of the RNA backbone). Diamonds describe the results of chemical probing (indicative of the exposure of the bases; D- DMS modifications; C- CMCT modifications; K- kethoxal modifications). Triangles describe the results of enzymatic probing (A- RNase A; V- RNase V1). Empty red diamonds or triangles correspond to weaker (1.5× to 4.5×) protection; filled red diamonds or triangles correspond to stronger (5× and up) protection. The diagrams and nomenclature of the structural elements are generally based on the work of Frank et al. (2000), Li et al. (2002), and Walker and Avis (2004) with minor corrections (Materials and Methods).
FIGURE 3.
FIGURE 3.
The protein components of RNase MRP/ RNase P preparations. This is a 15% SDS-polyacrylamide gel stained with Coomassie blue.

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