CHARACTERIZATION OF THE ESCHERICHIA-COLI RNA 3'-TERMINAL PHOSPHATE CYCLASE AND ITS SIGMA(54)-REGULATED OPERON

The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conv ersion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the e nd of various RNA substrates, Recent cloning of a cDNA encoding the hu man cyclase indicated that genes encoding cyclase-like proteins are co nserved among Eucarya, Bacteria, and Archaea, The protein encoded by t he Escherichia coli gene was overexpressed and shown to have the RNA 3 '-phosphate cyclase activity (Genschik, P,, Billy, E,, Swianiewicz, M, , and Filipowicz, W, (1997) EMBO J. 16, 2955-2967), Analysis of the re quirements and substrate specificity of the E. coli protein, presented in this work, demonstrates that properties of the bacterial and human enzymes are similar, ATP is the best cofactor (K-m = 20 mu M), wherea s GTP (K-m = 100 mu M) and other nucleoside triphosphates (NTPs) act l ess efficiently, The enzyme undergoes nucleotidylation in the presence of [alpha-P-32]ATP and, to a lesser extent, also in the presence of o ther NTPs. Comparison of 3'-phosphorylated oligoribonucleotides and ol igodeoxyribonucleotides of identical sequence demonstrated that the la tter are at least 300-fold poorer substrates for the enzyme. The E. co li cyclase gene, named rtcA, forms part of an uncharacterized operon c ontaining two additional open reading frames (ORFs), The ORF positione d immediately upstream, named rtcB, encodes a protein that is also hig hly conserved between Eucarya, Bacteria, and Archaea. Another ORF, cal led rtcR, is positioned upstream of the rtcA/rtcB unit and is transcri bed in the opposite direction, It encodes a protein having features of sigma(54)-dependent regulators. By overexpressing the N-terminally tr uncated form of RtcR, we demonstrate that this regulator indeed contro ls expression of rtcA and rtcB in a sigma(54)-dependent manner, Also c onsistent with the involvement of sigma(54) the region upstream of the transcription start site of the rtcA/rtcB mRNA contains the -12 and - 24 elements, TTGCA and TGGCA, respectively, characteristic of sigma(54 )-dependent promoters. The cyclase gene is nonessential as demonstrate d by knockout experiments, Possible functions of the cyclase in RNA me tabolism are discussed.