The process of tRNA maturation requires extensive posttranscriptional
modification. These modifications include 5’ leader removal, 3’ CCA addition,
intron splicing, and extensive base modification. The enzyme responsible for the
removal of the 5’ leader is known as Ribonuclease P (RNase P). This
ribonucleoprotein complex is present in all cells and cellular compartments that
perform translation. In this dissertation, the archaeal Ribonuclease P protein
aRpp29 from Archaeoglobus fulgidus was structurally characterized using
nuclear magnetic resonance (NMR) and X-ray crystallography techniques. The structure of aRpp29 consists of an amino terminal α-helix followed by
a six-stranded, anti-parallel β-sheet and then an α-helix at the carboxy terminus.
The three dimensional structure forms a semi-closed barrel, wrapped around a
well-conserved hydrophobic core. The α-helices align in an anti-parallel
orientation, capping the open end of the structure. There are well-conserved
charged residues that may present a surface for interactions with either the
RNase P RNA or the substrate tRNA. An interesting feature of this structure is an
internal salt bridge formed by a triad of conserved residues. This feature may
confer the unusual stability observed over a wide range of pH and temperatures.
The investigation of the structure of aRpp29 using NMR revealed distinct
differences when compared to the structure solved using X-ray crystallography.
The solution structure forms the same six stranded anti-parallel β-sheet but lacks
stable amino and carboxy terminal helices, indicating that ~40% of the protein is
in an equilibrium between a folded and unfolded state. This finding was further
investigated by measuring circular dichroism and amide proton exchange rates.
The structure of aRpp29 reveals that it is a variant of the Sm-fold (or likeSM) family of proteins. These proteins are involved in a variety of processes
involving RNA, including splicing and transcriptional regulation. Sm proteins and their homologs form heptameric rings in solution, although untracentrifugation
studies show that aRpp29 forms a monomer in solution.
The structural studies of archaeal Ribonuclease P protein Rpp29
presented in this dissertation provide an essential step toward understanding the
overall architecture of ribonuclease P.Institute for Cellular and Molecular Biolog
