Helicase Activity Plays a Crucial Role for RNase R Function in Vivo and for RNA Metabolism

Abstract

RNase R is a 3' to 5' hydrolytic exoribonuclease that has the unusual ability to digest highly structured RNA. The enzyme possesses an intrinsic, ATP-dependent RNA helicase activity that is essential in vitro for efficient nuclease activity against double-stranded RNA substrates, particularly at lower temperatures, with more stable RNA duplexes, and for duplexes with short 3' overhangs. Here, we inquired whether the helicase activity was also important for RNase R function in vivo and for RNA metabolism. We find that strains containing a helicase-deficient RNase R due to mutations in its ATP-binding Walker motifs exhibit growth defects at low temperatures. Most importantly, cells also lacking polynucleotide phosphorylase (PNPase), and dependent for growth on RNase R, grow extremely poorly at 34, 37, and 42 °C and do not grow at all at 31 °C. Northern analysis revealed that in these cells, fragments of 16S and 23S rRNA accumulate to high levels, leading to interference with ribosome maturation and ultimately to cell death. These findings indicate that the intrinsic helicase activity of RNase R is required for its proper functioning in vivo and for effective RNA metabolism.

Keywords: Escherichia coli (E. coli); RNA degradation; RNA helicase; bacteria; ribonuclease; ribosomal ribonucleic acid (rRNA) (ribosomal RNA).

FIGURE 1.

Northern analysis of RNA from MG1655* I⁻ and mutant derivatives lacking RNase R, PNPase, and the RNase R helicase. Cultures of the indicated strains were grown in LB medium to an A₆₀₀ = 0.6. Total RNA was isolated and resolved in a 1.2% agarose gel. A, cultures were grown at 37 °C; gels were stained with ethidium bromide. L denotes RNA ladder. B, cultures were grown at 37 °C to an A₆₀₀ = 0.6, and then shifted to 31 °C for 4 h; gels were stained with ethidium bromide. C, Northern analysis using probes specific for 16S and 23S rRNA of the gel shown in panel A. D, Northern analysis using probes specific for 16S and 23S rRNA of the gel shown in panel B. Arrows indicate the positions of the major rRNA fragments. Lines and numbers above the gels indicate the positions of the probes on the 16S and 23S RNA sequences.

FIGURE 2.

Growth and complementation analysis of stains lacking the RNase R helicase activity or DEAD-box RNA helicases. Cells were grown on LB plates at the temperatures and for the times indicated. A, growth of E. coli Mg1655*, Mg1655*Δrnr, Mg1655*Δ6helicase, Mg1655*Δrnr,Δ6helicase, and Mg1655*Δrnr,Δ6helicase containing pET44rnr was measured. B, complementation of Mg1655*Δrnr,Δ6helicase by pET44 containing WT rnr or mutated rnr genes. Leaky expression of the inserted genes in the plasmid system was used; isopropyl-1-thio-β-d-galactopyranoside was not added to the plates.