Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA.
Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.
Lambert
Meredith Newby
MN
Department of Chemistry, University of Michigan, Ann Arbor, 48109-1055, USA.
Hoerter
John A H
JA
Pereira
Miguel J B
MJ
Walter
Nils G
NG
D020029:Base Pairing; D001483:Base Sequence; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012269:Ribosomal Proteins; D012270:Ribosomes / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry / Q000378:metabolism*
D002712:Chlorides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; D012996:Solutions; C023509:ribosomal protein S16; D013725:Terbium; C007936:hexaminecobalt (III) trichloride; D003035:Cobalt; D008274:Magnesium