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Appl Environ Microbiol. 2011 Nov;77(22):7896-904. doi: 10.1128/AEM.05683-11. Epub 2011 Sep 16.

rnr gene from the antarctic bacterium Pseudomonas syringae Lz4W, encoding a psychrophilic RNase R.

Author information

1
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.

Abstract

RNase R is a highly processive, hydrolytic 3'-5' exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacterium Pseudomonas syringae Lz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His₆-tagged form of the P. syringae RNase R (RNase R(Ps)), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg²⁺ and Mn²⁺) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase R(Ps) exhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structured malE-malF RNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5'-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain by Escherichia coli RNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase R(Ps) in vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase R(Ps) activity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.

PMID:
21926201
PMCID:
PMC3208988
DOI:
10.1128/AEM.05683-11
[Indexed for MEDLINE]
Free PMC Article

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