• Title, Summary, Keyword: Double-stranded RNA binding factor

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RNA Binding Specificities of Double-Stranded RNA Binding Protein (RBF) as an Inhibitor of PRK Kinase (PKR인산화효소 억제인자인 이중선RNA결합단백질 (RBF)의 RNA결합특이성)

  • 박희성;최장원
    • Journal of Life Science
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    • v.6 no.4
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    • pp.234-240
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    • 1996
  • A double-stranded RNA binding factor (RBF), characterized as an inhibitor of PKR kinase in our previous study, was evaluated for its RNA binding specificities by RNA gel electrophoretic mobility shift analysis and membrane filter binding assay, RBF displayed affinities for a broad range of RNAs including viral RNAs and synthetic RNAs consiting of stem and loop structures. GC-rich RNA stem helices as short as 11 bp are suggested to represent the minimal binding motif for RBF. RBF binding to all the natural RNAs tested was reversible by poly(I): poly(C) addition, but E. coli 5S RNA was inefficient.

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Isolation of cDNA Encoding Double-Stranded RNA Binding Protein (RBFII) (이중선RNA결합담백질(RBFII)의 cDNA분리)

  • 박희성
    • Journal of Life Science
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    • v.7 no.3
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    • pp.167-171
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    • 1997
  • As an initial effort to elucidate RNA: protein binding in a way to regulate translation initiation and phosphorylation, a cDNA encoding a double-stranded RNA binding factor (RBFII)was isolated from Hela ZAPII cDNA library by affinity screening using [$\alpha$$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA. The nucleotide sequence of RBF (or TRBP) cDNA except the 5’end. At the 5’end, This common ORF was fused in-frame to N-terminal residues of Lac-Z through a unique 138 nt sequence encoding 46residues in the case of RBFII and a 63 nt sequence encoding 21 residuces in the case of RBFI. The context of ATG appearing first in the sequences suggests that both these cDNA inserts are incomplete at the 5’end.

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Comparative Study of Nucletic Acid Binding of the Purified RBF Protein and Its Inhibition of PKR phosphorylation (RBF정제단백질의 핵산결합도 및 PKR효소의 인산화억제효과의 비교에 관한 연구)

  • 박희성;김인수
    • Journal of Life Science
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    • v.8 no.2
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    • pp.119-125
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    • 1998
  • Column-purified double-stranded RNA binding factor (RBF) protein was tested for its binding affinity for the different forms of nucleic acids structure such as single-stranded(ss) and double-stranded(ds)RNA and ss- and dsDNA. The RBF protein was incubated with each of these nucleic acid structures in separate reactions and its comparative binding affnity was visualized by SDS-polyacrylamide gel electrophoresis. The RBF protein bound to the dsRNA molecule to form a tight RNA:protein complex in agreement with previous studies, but not to the other nucleic acid molecules confirming its distinctive affinity for the dsRNA structure. In phosphorylation assay in vito, the purified RBF protein significantly inhibited the autophosphorylation of the PKR derived from not only human but mouse source in the presence of poly(I):poly(C). It is suggesting that PKR vs. RBF is similarly under a competitive interaction among different eukaryotic organisms during protein synthesis.

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Mutational Analyses of Translation Initiation Factor eIF1A in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 번역 개시 인자 eIF1A 돌연변이에 대한 분석)

  • Kwon, Sung-Hun;Kim, Jun-Ho;Choi, Bo-Kyung;Kim, Na-Yeon;Choi, Do-Hee;Park, Kyoung-Jun;Eoh, Jung-Hyun;Bae, Sung-Ho
    • Korean Journal of Microbiology
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    • v.45 no.3
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    • pp.239-245
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    • 2009
  • Translation initiation factor eIF1A performs essential functions in various initiation steps including 43S preinitiation complex formation in eukaryotes, and contains a highly conserved oligonucleotide-binding (OB) fold. In our previous study, we discovered that eIF1A possesses RNA annealing activity and forms a stable complex with double-stranded RNA. In this study, we initiated site-directed mutations in eIF1A to find the active sites for these biochemical activities and to investigate whether they are essential functions for yeast cell growth. A truncated protein, eIF1A($\Delta$T), devoid of both N- and C-terminal domains but containing an intact OB-fold exhibited RNA annealing activity. In contrast, all point mutations in OB-fold domain, except R57D, impaired both RNA annealing and dsRNA binding activities, indicating that the intact OB-fold domain is required for both activities. Viabilities of the mutant yeast cells were not correlated with RNA annealing activity but with the in vivo protein stabilities in the case of R57D and K94D.

Function of mORF1 Protein as a Terminal Recognition Factor for the Linear Mitochondrial Plasmid pMLP1 from Pleurotus ostreatus

  • Kim, Eun-Kyoung;Roe, Jung-Hye
    • Journal of Microbiology
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    • v.37 no.4
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    • pp.229-233
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    • 1999
  • The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

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