title: Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe.
abstract: Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques. Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions. Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid. Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript. Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms. Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site. The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM). Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule. This conclusion is supported by the known biological activity of the E. coli tRNAPhe gene transcript.
authors.affiliation: Engelhardt Institute of Molecular Biology, Moscow 117984, Russia.
authors.forename: V
authors.initials: V
authors.lastname: Serebrov
authors.forename: K
authors.initials: K
authors.lastname: Vassilenko
authors.forename: N
authors.initials: N
authors.lastname: Kholod
authors.forename: H J
authors.initials: HJ
authors.lastname: Gross
authors.forename: L
authors.initials: L
authors.lastname: Kisselev
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli; D005456:Fluorescent Dyes; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015125:Oxyquinoline / Q000031:analogs & derivatives; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry
chemical_list: D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D015125:Oxyquinoline; C013053:8-hydroxyquinoline-5-sulfonic acid; D008274:Magnesium