Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA.
Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.
Samaha
R R
RR
Sinsheimer Laboratories, University of California, Santa Cruz 95064.
O'Brien
B
B
O'Brien
T W
TW
Noller
H F
HF
D001483:Base Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D012336:RNA, Ribosomal, 16S / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D012336:RNA, Ribosomal, 16S; D012261:Ribonucleoproteins