RNase P                                        "Why use Proteins to do a Nucleotides job?"

 

 

The long term goal of this research project is to further our understanding of (1) the mechanisms of catalysis used by ribozymes as compared to protein enzymes, and (2) the structure of RNA binding proteins and protein/RNA complexes.  To this end, we have begun investigating the enzymology and structural biology of the catalytic ribonucleoprotein RNase P.  This novel catalyst contains an RNA subunit (P RNA) and a protein subunit (P protein).  As of date, we solved the first structure of the protein component of ribonuclease P in a collaboration with the Christianson laboratory at UPenn and demonstrated that one of the functions of this protein is to bind the leader sequence of the precursor-tRNA substrate.  This  

Secondary structure model of P RNA 

Kurz, J., and Fierke, C. A. Ribonuclease P: a ribonucleoprotein enzyme.  (2000) Current Opinion in  Chemical Biology. 4,  553-558.

function places the protein component in the proximity of the active site and suggests that the active site occurs at the RNA/protein interface.  This is the first case where the RNA and protein components both contribute to the molecular recognition properties of an enzyme.  Furthermore, the positions of key metal ions have been identified using phosphorothioate substitution studies.

      We have further investigated the structure of the RNase P protein and complexes of the protein with precursor tRNA segments and P RNA by utilizing both UV-crosslinking and chemical affinity to enhance low resolution models of the biologically active complex for comparison with the high resolution structures.  We have initiated the kinetic analysis, using both radiological and fluorescent assays (in conjunction with the Dr. Nils Walter's lab here at U of M), of wild-type and variant forms of both the P protein and P RNA subunits for the systematic dissection of the mechanism of catalysis employed by RNase P.  We have an on-going collaboration with Dr. David Engelke's lab here at U of M to probe RNA-protein interactions within the more complex S. cerevisiae RNase P holoenzyme.  Recently, we have started to probe the mechanism of catalysis of the mitochondrial RNase P, a putative protein-only complex.

Recent Publications     

  1. Crary SM, Kurz JC, Fierke CA. Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P. (2002) RNA.  7, 933-47.
  2. Kurz JC, Fierke CA. The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit. (2002) Biochemistry. 41, 9545-58.
  3. Henkels CH, Kurz JC, Fierke CA, Oas TG.  Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein. (2001) Biochemistry. 9, 2777-89.
  4. Ziehler WA, Day JJ, Fierke CA, Engelke DR.  Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P. 2000
    Biochemistry    . 32, 9909-16.

PubMed Search (most recent publications)

Project Researchers
Post Doctoral Fellow
Dr. John Hsieh

Graduate Students
J. Kristin Smith Koutmou
Wan Lim
Xin Liu

Questions about this project? Email Kristin: jkristin at umich.edu

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