Description:
<jats:title>ABSTRACT</jats:title>
<jats:p>
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
<jats:named-content content-type="genus-species">Pseudomonas syringae</jats:named-content>
Lz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (
<jats:italic>rnr</jats:italic>
) locus from this bacterium. By cloning and expressing a His
<jats:sub>6</jats:sub>
-tagged form of the
<jats:named-content content-type="genus-species">P. syringae</jats:named-content>
RNase R (RNase R
<jats:sup>Ps</jats:sup>
), 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 (Mg
<jats:sup>2+</jats:sup>
and Mn
<jats:sup>2+</jats:sup>
) 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
<jats:sup>Ps</jats:sup>
exhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structured
<jats:italic>malE-malF</jats:italic>
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
<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>
RNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase R
<jats:sup>Ps</jats:sup>
<jats:italic>in vitro</jats:italic>
. The ability of the nonhydrolyzable ATP-γS to inhibit RNase R
<jats:sup>Ps</jats:sup>
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.
</jats:p>