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

Abstract

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 transsplicing 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 ϳ122-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.The Trypanosomatidae are an important family of flagellated, unicellular organisms that parasitize a diverse array of multicellular organisms. The disease-causing trypanosomes, found primarily in developing countries, inflict debilitating symptoms and eventual death on many thousands of people annually.A distinguishing feature of the protozoan family Trypanosomatidae is the presence of a capped, 39-nucleotide (nt) 1 RNA preceding the translational start site of every mRNA (reviewed in Ref. 1). This short RNA is derived from a 3-4-fold longer RNA, called the spliced leader (SL) RNA or Mini Exon Donor RNA. The 5Ј-end of the SL RNA fuses to a primary mRNA and the 3Ј-end is rapidly degraded. Although the trans-splicing of mRNA by an SL RNA occurs in several trematodes, nematodes, and euglenoids, only in trypanosomatids is addition of an SL RNA essential for the formation of every mRNA (2).The SL RNA genes are members of the small nuclear (sn) RNA class of eukaryotic genes (3-5). These genes encode short, nonpolyadenylated RNAs that participate in mRNA-processing reactions. In higher eukaryotes, in which snRNA genes have been extensively studied, snRNAs are synthesized from independent transcription units that contain promoter elements specific to this gene class (6, and reviewed in Refs. 7-9). The presence of common promoter elements was unexpected since some snRNA genes are transcribed by RNA polymerase II and others by RNA polymerase III. In trypanosomatids, the SL RNA genes appear to be transcribed by RNA polymerase II, whereas other snRNA genes, including the U2 snRNA, U6 snRNA, and U3 snRNA homolog (U-snRNA B) genes, are transcribed by RNA polymerase III (5, 10 -12).To characterize transcription of the SL RNA, we began by assessing the effects of mutations on a marked copy of an SL RNA gene. The marked gene was reintr...

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

“…Similar studies using increasing amounts of either the individual PBP-1 (lanes 8 -10) or PBP-2 (lanes 5-7) binding sites also reduced transcription efficiencies. PBP-2E was less efficient in blocking expression than was PBP-1E, which is consistent with our observation that PBP-2 alone binds DNA poorly (15). Adding the Inr t region to the transcription reaction stopped transcription from template DNA, suggesting that a trans-acting Inr t -binding protein is being sequestered (Fig.…”

“…PBP-1 and PBP-2 Proteins Are Important for SL RNA Transcription in Vitro-The requirement for PBP-1E and PBP-2E to be within 20 nt of each other is consistent with the previous gel mobility shift data in which PBP-1 and PBP-2 were required to form stable complexes at the SL RNA gene promoter (15). To assess directly the role of PBP-1 and PBP-2 in SL RNA transcription we performed protein sequestration and subsequent add-back experiments using in vitro transcription assays.…”

“…The P400 fraction was prepared by dialyzing the nuclear extract (ϳ100 mg of protein) against buffer 2 at 50 mM potassium glutamate and loading onto a 5-ml phosphocellulose (P-11, Whatman) column as described (15). Proteins were eluted using a step gradient (200, 400, 600 mM potassium glutamate) in buffer 2.…”

“…The specific DNA affinity-purified PBP-1 and PBP-2 proteins were prepared as described previously (15) and dialyzed against buffer 2 at 20 mM potassium glutamate (the final protein concentration was ϳ1.6 mg/ml) before addition to the transcription reaction. For the sequestration experiments, the acetylated bovine serum albumin was from New England BioLabs, Inc.…”

“…In a dearth of any previously defined trypanosome transcription factors, PBP-1 and PBP-2 (15), which are sequence-specific DNA-binding proteins initially identified in our laboratory, emerge as the first proteins that function to promote efficient SL RNA transcription. These studies also reveal that correct 5Ј-end formation of the SL RNA, which is crucial to proper capping of the SL, is dependent on the presence of a 5-bp element, the trypanosome initiator (Inr t ).…”