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.

Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

“…The possibility of ineffective ribosome assembly should be considered since Krzyzosiak et al (1987) noted that in vitrosynthesized G1400 containing rRNA is inefficient in the assembly process. However, another publication from the same group showed that such G1400 containing rRNA could be assembled efficiently provided that higher rRNA concentrations were used in the assembly reaction.…”

Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

“…The overall scheme involves the assembly of a mutant ribosome pool in vivo, thus avoiding the inefficiency of in vitro reconstitution (22)(23)(24)(25)(26). The pool of variant ribosomes isolated from Escherichia coli is then subjected to selective pressure in vitro, where the definition of ''functional'' can be exquisitely manipulated.…”

Purification, Cloning, and Characterization of the 16 S RNA m2G1207 Methyltransferase from Escherichia coli

“…In rRNA, which next to tRNA contains the widest known assortment of modified nucleosides, no function has so far been ascribed to them, although a potential role in peptide bond formation for a subset of the modified nucleosides of Escherichia coli 23 S RNA has been postulated (3). In E. coli 16 S RNA, in which all of the modified nucleosides are known and their locations precisely determined, none was essential for any of the known ribosomal functions, although ribosomal efficiency was reduced to approximately half when all were absent (4,5). Evidence has been presented that this lowered efficiency of the 30 S subunits may be attributable to the lack of one or more of the 10 methylated residues and/or the single pseudouridine (6) in the RNA (7).…”