Arun Malhotra

Tristan J. Fiedler

The Scientific World Journal

English

Fiedler, Tristan J

Malhotra, Arun

University of Miami: Scholarship Miami

The Scientific World JOURNAL

Crystallographic Studies of Bacterial Exoribonucleases

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Advances in Gene Technology: The Genome and Beyond – 
Structural Biology for Medicine (Proceedings of the 2002 Miami 
Nature Biotechnology Winter Symposium) 
TheScientificWorld 2002, 2(S2), 53–54 
ISSN 1532-2246; DOI 10.1100/tsw.2002.27 
  
  
  
  
CRYSTALLOGRAPHIC STUDIES OF BACTERIAL EXORIBONUCLEASES 
  
Tristan J. Fiedler and Arun Malhotra
Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, PO 
Box 016129, Miami, FL 33101-6129 
  
INTRODUCTION. Ribonucleases (RNases) play a central role in all cellular RNA processes. 
These processes include mRNA degradation, and maturation and turnover of stable RNAs, which 
are vital for the proper functioning of all cells. E. coli has served as a model system for 
understanding the role of ribonucleases in RNA metabolism, and eight distinct exoribonucleases 
have been identified in this bacterium. Of these, three (RNase T, RNase D, and oligoribonuclease) 
are members of a larger exonuclease superfamily (named the DEDD exonuclease family, after the 
four invariant acidic residues in these proteins) that includes the proof-reading domains of DNA 
polymerases[1]. 
  
While these three proteins share similar sequence motifs, they are functionally quite different. 
RNase T is involved in end-turnover of tRNAs and the maturation of tRNAs, 23S, and 5S RNAs. 
Interestingly, RNase T has been shown recently to also be a potent Dnase[2], and may play a role 
in end-trimming reactions during DNA recombination and repair[3]. RNase D is also involved in 
the maturation of tRNAs and small RNAs, but mainly as a backup enzyme. RNase D functions as a 
monomer, unlike RNase T and oligoribonuclease that exist as dimers. Oligoribonuclease catalyzes 
the degradation of very short RNAs (4 residues or shorter). It is the only exoribonuclease essential 
for cell viability, and its absence leads to accumulation of small oligoribonucleotides derived from 
mRNAs[4].  
  
We have initiated crystallographic studies of this family of bacterial exoribonucleases, in 
collaboration with the laboratory of Dr. Murray Deutscher at the University of Miami, to 
structurally characterize these proteins and understand their mechanisms of exoribonuclease 
activity at an atomic resolution. The long term goals of this research are to understand the 
structures and mechanisms of action of all the exoribonucleases in a single organism (E. coli); 
these studies will complement a parallel study to completely determine and characterize the 
physiological role of all the exoribonucleases in E. coli, now underway in the Deutscher 
laboratory. 
  
METHODS. RNase T and oligoribonuclease were overexpressed and purified from E. coli using 
standard column chromatographic techniques. RNase T was crystallized using ammonium sulfate 
as a precipitant. Oligoribonuclease was crystallized using sodium citrate as a precipitant, using the 
hanging drop methodology. Selenomethionine labeled oligoribonuclease was also purified, and 
crystallized for MAD phasing. These crystals are in the orthorhombic spacegroup (P212121; a = 
70.4, b = 72.9, c = 147.8 Å). 
  
Diffraction data on oligoribonuclease were collected to 2.0 Å resolution on the X8c and X12b 
beamlines at the National Synchrotron Light Source, Brookhaven National Laboratory. For the 
selenomethionine labeled oligoribonuclease, we collected diffraction data at three wavelengths for 
multiple anomalous diffraction (MAD) phasing. Twenty SeMet sites (five in each of the four 
oligoribonuclease molecules) were located using SOLVE[5], and used to obtain an experimental 
map extending to about 2.2 Å. Model building is currently in progress. 
  
RESULTS. We are currently building an atomic model for oligoribonuclease using experimental 
phases extending to 2.2 Å. Two dimers of oligoribonuclease can be seen in the asymmetric unit. 
The atomic structure and detailed structural analysis for this protein will be presented. 
  
ACKNOWLEDGEMENT. This work was supported by a Florida Biomedical Research 
Foundation Award #BM030 (to A.M.). 
  
REFERENCES 
1. Zuo, Y. and Deutscher, M.P. (2001) Nucl. Acids Res. 29, 1–10. 
2. Zuo, Y. and Deutscher, M.P. (1999) Nucl. Acids Res. 27, 4077–4082. 
3. Ghosh, S. and Deutscher, M.P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4372–
4377. 
4. Viswanathan, M., Lanjuin, A., and Lovett, S.T. (1999) Genetics 151, 929–934. 
5. Terwilliger, T. and Berendzen, J. (1999) Acta Crystallogr. D55, 849–861. 
  
 
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Crystallographic Studies of Bacterial Exoribonucleases

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