Inactivation of the RluD Pseudouridine Synthase Has Minimal Effects on Growth and Ribosome Function in Wild-TypeEscherichia coliandSalmonella enterica

Abstract

The Escherichia coli rluD gene encodes a pseudouridine synthase responsible for the pseudouridine (⌿) modifications at positions 1911, 1915, and 1917 in helix 69 of 23S rRNA. It has been reported that deletion of rluD in K-12 strains of E. coli is associated with extremely slow growth, increased readthrough of stop codons, and defects in 50S ribosomal subunit assembly and 30S-50S subunit association. Suppressor mutations in the prfB and prfC genes encoding release factor 2 (RF2) and RF3 that restore the wild type-growth rate and also correct the ribosomal defects have now been isolated. These suppressors link helix 69 ⌿ residues with the termination phase of protein synthesis. However, further genetic analysis reported here also reveals that the slow growth and other defects associated with inactivation of rluD in E. coli K-12 strains are due to a defective RF2 protein, with a threonine at position 246, which is present in all K-12 strains. This is in contrast to the more typical alanine found at this position in most bacterial RF2s, including those of other E. coli strains. Inactivation of rluD in E. coli strains containing the prfB allele from E. coli B or in Salmonella enterica, both carrying an RF2 with Ala246, has negligible effects on growth, termination, or ribosome function. The results indicate that, in contrast to those in wild bacteria, termination functions in E. coli K-12 strains carrying a partially defective RF2 protein are especially susceptible to perturbation of ribosome-RF interactions, such as that caused by loss of h69 ⌿ modifications.Pseudouridine (⌿) is among the most common posttranscriptional modifications occurring in rRNA and has been found at conserved regions of the rRNAs in organisms from all three kingdoms (28,29). In Escherichia coli, there are 11 ⌿ residues in 16S and 23S rRNAs, and the 7 synthases responsible for these modifications have now been identified (30). The functions of specific ⌿ residues have been investigated by deletion of the corresponding ⌿ synthase genes (rsuA, rluA, rluB, rluC, rluD, rluE, and rluF) and characterization of the resulting deletion mutants. With the notable exception of rluD, deletion of individual ⌿ synthase genes has little effect on cell growth (29). In contrast, inactivation or deletion of rluD has been reported to have profound effects on cell growth, 50S subunit assembly, and subunit association (14, 32). RluD is responsible for ⌿1911, ⌿1915, and ⌿1917 modifications in helix 69 (h69) of 23S rRNA. This functionally important stemloop structure forms a bridge between the 30S and 50S ribosomal subunits and interacts with translation factors and tRNAs at multiple stages of protein synthesis (39).The location of ⌿ residues in h69 and the phenotypes of the ⌬rluD mutants lacking h69 ⌿ residues suggest that these posttranscriptional modifications are important for ribosome function. Strains lacking RluD are extremely unstable and rapidly give rise to faster-growing, suppressor-containing derivatives (12,14). Some insights into h69 ⌿ functions were...

Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

“…E. coli MC323 (F Ϫ Ϫ lacZ521 [Tn10 linked] rph-1) was the host strain used for recombineering and screening for accuracy-altering mutations. MC361 [F Ϫ ara ⌬(gpt-lac)5 thi prfB (from E. coli B)] carries the fully active A246 RF2 allele (16) and was used for reconstruction of the selected mutants. Plasmids pKD46, pKD4, and pCP20 were used in recombineering (17).…”

Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

“…Inactivation of rluD encoding pseudouridine synthase was initially shown to have a negative effect on E. coli growth and ribosome function; however, a recent study demonstrated that rluD mutants are unstable and quickly give rise to suppressors (O'Connor & Gregory, 2011). The rluD suppressor mutations were found in pfrB and prfC encoding release factor 2 (RF2) and release factor 3 (RF3), respectively (O'Connor & Gregory, 2011).…”

“…The rluD suppressor mutations were found in pfrB and prfC encoding release factor 2 (RF2) and release factor 3 (RF3), respectively (O'Connor & Gregory, 2011). The investigation of unknown essential genes, particularly assigning function to the short ORFs encoding essential proteins of 50 or fewer amino acids, is challenging.…”

Comprehending a Molecular Conundrum: Functional Studies of Ribosomal Protein Mutants from Salmonella enterica Serovar Typhimurium

“…Mutations in the genes prmB (encoding class-I release factor RF2) and rpsG (encoding ribosomal protein S7) represent two well-characterized examples. O' Connor et al have reported that in all K-12 strains of E. coli, RF2 carries a threonine residue at position 246 as opposed to alanine, which is typically found at this position in the majority of bacterial RF2 proteins (O'Connor & Gregory, 2011). In the case of rpsG, a point mutation alters its canonical UGA stop codon to a leucine codon (Schaub & Hayes, 2011).…”