PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi.
In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli, showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.
Wen
L M
LM
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678, USA.
Xu
P
P
Benegal
G
G
Carvalho
M R
MR
Buck
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C425292:PPB1 protein, Trypanosoma cruzi; D015800:Protozoan Proteins; D020038:RNA, Spliced Leader; D014157:Transcription Factors