Characterization of Two Protein Activities That Interact at the Promoter of the Trypanosomatid Spliced Leader RNA

• Published in: The Journal of biological chemistry Vol. 272; no. 52; pp. 33344 - 33352
• Main Authors: Luo, Hua, Bellofatto, Vivian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-12-1997; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; Deoxyribonuclease I - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Leishmania - chemistry; Leishmania - genetics; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Promoter Regions, Genetic; Protozoan Proteins - chemistry; Protozoan Proteins - isolation & purification; Protozoan Proteins - metabolism; RNA Splicing; RNA, Messenger - genetics; RNA, Protozoan - genetics; Transcription Factors - chemistry; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.52.33344

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 ∼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.