Interactions of a didomain fragment of the Drosophila sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions.
Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.
Kim
I
I
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Japan.
Muto
Y
Y
Watanabe
S
S
Kitamura
A
A
Futamura
Y
Y
Yokoyama
S
S
Hosono
K
K
Kawai
G
G
Takaku
H
H
Dohmae
N
N
Takio
K
K
Saskamoto
H
H
Shimura
Y
Y
D000818:Animals; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D007301:Insect Hormones / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011685:Purine Nucleotides / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014529:Uridine / Q000737:chemistry / Q000378:metabolism
D029721:Drosophila Proteins; D007301:Insect Hormones; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D011685:Purine Nucleotides; D012322:RNA Precursors; D016601:RNA-Binding Proteins; C067777:Sxl protein, Drosophila; C097162:Tra protein, Drosophila; D003903:Deuterium; D014529:Uridine