Characterization of two protein activities that interact at the promoter of the trypanosomatid spliced leader RNA.
All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in Leptomonas seymouri is a member of the small nuclear RNA gene family. However, the SL RNA is required in stoichiometric amounts for trans-splicing during mRNA formation. Expression of the SL RNA gene requires sequence elements at bp -60 to -70 and bp -30 to -40 upstream from the transcription initiation site. Using conventional and affinity chromatography, we have identified and characterized an approximately 122-kDa protein, promoter-binding protein (PBP) -1, that binds to double-strand DNA. The PBP-1-binding site is within the bp -60 to -70 element determined by DNase I footprinting. Therefore, PBP-1 is the first characterized double-strand DNA binding activity that interacts with a trypanosome gene promoter. A second protein, PBP-2, interacts with the PBP-1:DNA complex and its DNase I footprint extends to include the second promoter element (bp -30 to -40). An alteration of the spacing between the two promoter elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 are components of a transcription initiation complex that assembles within the SL RNA gene promoter.
Luo
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Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103, USA.
Bellofatto
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D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D007891:Leishmania / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D016053:RNA, Protozoan / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C400747:PBP-1 protein, Leptomonas seymouri; C400748:PBP-2 protein, Leptomonas seymouri; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D014157:Transcription Factors; D003850:Deoxyribonuclease I