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Abstract
Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.
Highlights
RNA maturation and decay pathways are fundamentally involved in gene expression and regulation in bacterial cells, and are defined by the coordinated action of endoribonucleases and exoribonucleases
RNase III is phosphorylated during T7 infection in a T7 protein kinase (T7PK)-dependent manner, and the catalytic activity of the enzyme increases ~4–fold upon phosphorylation in vivo[28]
T7PK phosphorylates serine in the α2-α3 loop of the RNase III catalytic domain
Summary
Phosphorylation received: 08 January 2016 accepted: 15 April 2016 Published: 06 May 2016. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. Double-helical structures that are formed by binding of small noncoding RNAs (sRNAs) provide RNase III targets, and regulate mRNA translation and/or stability[12,13]. Following T7 infection of Escherichia coli, the host RNA polymerase transcribes the phage early region, generating a ~7,000 nt polycistronic mRNA precursor that is processed co-transcriptionally by RNase III at five sites to provide the mature, optimally functional mono- and dicistronic mRNAs15,16. Expression of T7PK results in the phosphorylation of multiple cellular proteins, including translation factors and the β ′ subunit of the host RNA polymerase[21,22,23]. We present here an analysis of T7PK phosphorylation of RNase III, and describe the structural basis for the catalytic enhancement of dsRNA processing by phosphorylation
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