1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E.coli rho protein.
Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1-116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)10 to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1-130 of rho protein (Rho130) is found to be stably folded, both in absence and presence of nucleic acid. NMR studies of the complex of Rho130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho130 are similar to those of intact rho protein; therefore, Rho130 is a suitable model of the RNA-binding domain of Rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggests that Rho130 forms oligomers in the presence of longer oligonucleotides. 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.
Briercheck
D M
DM
Department of Biochemistry, University of Virginia, School of Medicine, Charlottesville 22908, USA.
Allison
T J
TJ
Richardson
J P
JP
Ellena
J F
JF
Wood
T C
TC
Rule
G S
GS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D009587:Nitrogen Isotopes; D012329:RNA, Bacterial; D012234:Rho Factor; D006859:Hydrogen