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The construction and analysis of lethal and second site suppressor mutations in two highly conserved regions of Escherichia coli 16S ribosomal RNA
Abstract
Mutagenized fragments of the 165 rRNA gene were placed within plasmid-borne rrnB operons and expressed under the control of the rrn promoters, P1P2. Several base changes within the conserved C1400 region led to reduced host-cell growth rate, and, notably, transition mutations at position 1395 or 1407, or the deletion of base 1400, appeared to be lethal. The latter mutations were investigated by transferring them to plasmids in which the P$\sb1$P$\sb2$ promoters had been replaced with the inducible leftward promoter of the phage lambda; it was found that host cells expressing the putative lethal mutations died within 3 to 4 generations. Analysis of the rRNA and ribosomes present at 3 generations after induction of the mutant operons demonstrated that the mutant rRNA was processed to 16S rRNA and that induction of the altered rRNA genes led to significant accumulation of free ribosomal subunits. Sequencing of the rRNA in 30S subunits and 70S ribosomes showed that mutant rRNA was assembled into subunits, but that the mutant subunits were assembled into 70S ribosomes at low frequency. Furthermore, subunits containing the altered 165 rRNA were less likely to associate with 50S subunits, in vitro, than wild-type subunits, suggesting that the lethal mutations led to defective subunit association, in vivo. In addition, the conserved nucleotide G1505 was mutagenized, and it was found that 3 point mutations at position 1505 could suppress the lethal phenotype associated with the U1395, U1407 or $\Delta$1400 mutations. The doubly-mutant rRNA appeared to be fully processed to 16S rRNA. Although the suppressed strains accumulated excess ribosomal subunits, 90% of the rRNA associated with both 30S and 70S ribosomal particles contained a mutant allele at position 1505 within 3 generations after induction. The latter observation is consistent with the ribosome-feedback model of rRNA transcription control, which posits that initiation-competent 70S ribosomes exert a negative feedback effect at the rrn P1P2 promoters. The data are interpreted as evidence that nonfunctional ribosomes carrying lethal mutations in the 1400 region can be rendered functional by second-site mutations at position 1505.
Subject Area
Molecular biology|Biochemistry|Cellular biology
Recommended Citation
Thomas, Cheryl Lynne, "The construction and analysis of lethal and second site suppressor mutations in two highly conserved regions of Escherichia coli 16S ribosomal RNA" (1991). Doctoral Dissertations Available from Proquest. AAI9132924.
https://scholarworks.umass.edu/dissertations/AAI9132924