Site-directed hydroxyl radical probing of 30S ribosomal subunits by using Fe(II) tethered to an interruption in the 16S rRNA chain.
Two in vitro transcripts, one corresponding to the 5' and central domains (residues 1-920) of 16S rRNA and the other corresponding to its 3' domain (residues 922-1542), assemble efficiently in trans with 30S ribosomal proteins to form a compact ribonucleoprotein particle that cosediments with natural 30S subunits. Isolated particles are similar in appearance to natural 30S subunits with electron microscopy and contain a full complement of the small subunit ribosomal proteins. The particles have a reduced ability to bind tRNA (attributable to the location of the discontinuity in a conserved region of the rRNA) near features that have been implicated in tRNA binding. Association of these two halves of 16S rRNA in trans must be stabilized by either previously unidentified RNA-RNA contacts or interactions mediated by ribosomal proteins because there are no known direct interactions between them. The trans construct was used to probe the three-dimensional RNA neighborhood around position 922 of 16S rRNA by generating hydroxyl radicals from Fe(II) tethered to the 5' end of the 3' transcript. Hydroxyl radical-induced cuts in the 16S rRNA chain were localized by primer extension to nucleotides 923-929 and 1192-1198, providing evidence for the mutual proximity of the 920 and 1192 regions.
Samaha
R R
RR
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.
Joseph
S
S
O'Brien
B
B
O'Brien
T W
TW
Noller
H F
HF
D002499:Centrifugation, Density Gradient; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D017665:Hydroxyl Radical / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000378:metabolism; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry*; D012343:RNA, Transfer / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000648:ultrastructure; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000648:ultrastructure; D014158:Transcription, Genetic / Q000235:genetics
D007502:Iron Chelating Agents; D009942:Organometallic Compounds; D012336:RNA, Ribosomal, 16S; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D012343:RNA, Transfer; D004492:Edetic Acid