Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding.
Recently, a novel uracil-DNA-degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil-DNA-degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA-UDE) revealed essential differences. UDE cannot be converted into RNA-UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real-time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA-assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA-dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA-processing enzyme.
Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Budapest, Hungary. bekesi@enzim.hu
Angela
A
Bekesi
Maria
M
Pukancsik
Peter
P
Haasz
Lilla
L
Felfoldi
Ibolya
I
Leveles
Villo
V
Muha
Eva
E
Hunyadi-Gulyas
Anna
A
Erdei
Katalin F
KF
Medzihradszky
Beata G
BG
Vertessy
D000818:Animals; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009843:Oligoribonucleotides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011679:Pupa / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D013696:Temperature; D044366:Transition Temperature
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011994:Recombinant Proteins; C519400:UDE protein, Drosophila; D012313:RNA; D004247:DNA; D012260:Ribonucleases; D010450:Endopeptidases