Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.
A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.
Valadão
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AF
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Minas Gerais 31270-010, Brazil.
Fantappie
M R
MR
LoVerde
P T
PT
Pena
S D J
SD
Rumjanek
F D
FD
Franco
G R
GR
D000818:Animals; D001426:Bacterial Proteins; D018509:DNA, Helminth / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000378:metabolism*; D015345:Oligonucleotide Probes; D017434:Protein Structure, Tertiary; D018510:RNA, Helminth / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012550:Schistosoma mansoni / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D018509:DNA, Helminth; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D015801:Helminth Proteins; D015345:Oligonucleotide Probes; D018510:RNA, Helminth; D011994:Recombinant Proteins; D014157:Transcription Factors; C109092:Y-box-binding protein 1, Schistosoma mansoni; C407094:cold shock protein, Bacillus