title: In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome.
abstract: Functional 30S ribosomes were reconstructed from total Escherichia coli 30S ribosomal proteins and 16S ribosomal RNA synthesized in vitro by T7 RNA polymerase. Up to 700 mol of RNA/mol of template could be obtained. The transcript lacked all ten normally modified bases and had three additional 5' G residues, an A----G change at position 2, and, in 22% of the molecules, one or two extra 3' residues. The synthetic 16S RNA could be assembled into a particle that cosedimented with authentic 30S and was indistinguishable from 30S by electron microscopy. When supplemented with the 50S subunit, the particles bound tRNA to the 70S P site in a codon- and Mg2+-dependent manner. The specific binding activity was 94% that of particles reconstituted with natural rRNA and 52% that of native 30S. Cross-linking to P site bound tRNA was also preserved. Changing C-1400, the residue known to be close to the anticodon of P site bound tRNA, to A had little effect on reconstitution, but the C----G substitution caused a marked inhibition of assembly. tRNA could bind to both reconstituted mutants, but cross-linking was greatly reduced. These results show that none of the modified bases of 16S RNA are essential for P site binding and that position 1400 may be more important for ribosome assembly than for tRNA binding. Base-specific in vitro mutagenesis can now be used to explore in detail the functional properties of individual residues in ribosomal RNA.
authors.forename: W
authors.initials: W
authors.lastname: Krzyzosiak
authors.forename: R
authors.initials: R
authors.lastname: Denman
authors.forename: K
authors.initials: K
authors.lastname: Nurse
authors.forename: W
authors.initials: W
authors.lastname: Hellmann
authors.forename: M
authors.initials: M
authors.lastname: Boublik
authors.forename: C W
authors.initials: CW
authors.lastname: Gehrke
authors.forename: P F
authors.initials: PF
authors.lastname: Agris
authors.forename: J
authors.initials: J
authors.lastname: Ofengand
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013604:T-Phages / Q000201:enzymology
chemical_list: D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D012321:DNA-Directed RNA Polymerases