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Molecular and Cellular Biology, July 2001, p. 4453-4459, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4453-4459.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins

Riaz Mahmood, Bidyottam Mittra, Jane C. Hines, and Dan S. Ray*

Molecular Biology Institute and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095-1570

Received 16 March 2001/Accepted 20 April 2001

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.

* Corresponding author. Mailing address: Molecular Biology Institute, University of California, 405 Hilgard Ave., Los Angeles, CA 90095-1570. Phone: (310) 825-4178. Fax: (310) 206-7286. E-mail: danray{at}mbi.ucla.edu.

Molecular and Cellular Biology, July 2001, p. 4453-4459, Vol. 21, No. 14
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.14.4453-4459.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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