Mutants with base changes at the 3'-end of the 16S RNA from Escherichia coli. Construction, expression and functional analysis

Abstract

The functionally important 3' domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid-encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U. Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the lambda PL promoter. In a strain coding for the temperature-sensitive lambda repressor cI857 the mutant RNAs could be expressed conditionally. We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees. The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3' domain of the 16S RNA for ribosomal function.

Amicoumacin A Inhibits Translation by Stabilizing mRNA Interaction with the Ribosome

“…The mutagenized plasmids were initially transformed in the POP2136 cells which were grown at 30°C to prevent expression of the mutant rrnB operon (Rottmann et al, 1988). Once mutant plasmids were identified, they were transformed into the SQ171Δ tolC (SQ171DTC) strain and the cells were then cured of wild type plasmid pCSacB by plating onto an LB/Amp plate supplemented with 5% sucrose (Zaporojets et al, 2003).…”

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA

“…Using this approach, we have managed to construct isogenic strains of E. coli that express either wild-type or anti-DB flip mutant rRNAs exclusively. A variety of previously studied, single-base alterations in helix 44 have been found to alter the ability of the mutant subunits to associate with 50S subunits (24)(25)(26). Although a very slight increase in the amount of free subunits was observed in sucrose gradients of anti-DB mutant ribosomes, cell lysates from strains expressing exclusively wild-type or the anti-DB flip mutant rRNAs displayed essentially identical levels of 70S ribosomes and polysomes on sucrose gradients.…”

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes

“…In prokaryotes, helix 47 has been found to undergo allosteric transitions upon binding of the 50S subunit (31) and both the nucleotides and the phosphate backbone of this region are protected from modification by the 50S subunit (3,6,29). Furthermore, removal of the helix or perturbation of its secondary structure by point-mutations abolishes or reduces 70S ribosome formation (32)(33)(34).…”