Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.
Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5' untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5' UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6x octamer) into the 5' UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6x octamer RNA and TOP2 5' UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6x octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6x octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.
Mahmood
R
R
Molecular Biology Institute and Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095-1570, USA.
Hines
J C
JC
Ray
D S
DS
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D002453:Cell Cycle / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D016831:Crithidia fasciculata / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D017930:Genes, Reporter; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism
D020121:5' Untranslated Regions; D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan