Abstract
The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of various RNA substrates. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the 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 requirements 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 (Km = 20 microM), whereas GTP (Km = 100 microM) and other nucleoside triphosphates (NTPs) act less efficiently. The enzyme undergoes nucleotidylation in the presence of [alpha-32P]ATP and, to a lesser extent, also in the presence of other NTPs. Comparison of 3'-phosphorylated oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are at least 300-fold poorer substrates for the enzyme. The E. coli cyclase gene, named rtcA, forms part of an uncharacterized operon containing two additional open reading frames (ORFs). The ORF positioned immediately upstream, named rtcB, encodes a protein that is also highly conserved between Eucarya, Bacteria, and Archaea. Another ORF, called rtcR, is positioned upstream of the rtcA/rtcB unit and is transcribed in the opposite direction. It encodes a protein having features of sigma54-dependent regulators. By overexpressing the N-terminally truncated form of RtcR, we demonstrate that this regulator indeed controls expression of rtcA and rtcB in a sigma54-dependent manner. Also consistent with the involvement of sigma54, 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 sigma54-dependent promoters. The cyclase gene is nonessential as demonstrated by knockout experiments. Possible functions of the cyclase in RNA metabolism are discussed.
Highlights
Known enzymes, among them many secretory degradative nucleases such as RNases A or T1, cyclic phosphates are formed as intermediates that are subsequently opened into 3Ј-phosphomonoesters [1, 2]
Requirements of the E. coli Cyclase—To compare the human and E. coli enzymes, we have studied requirements of the overexpressed and purified bacterial protein using oligoribonucleotides labeled at the 3Ј-terminal phosphate, AAAAUAAAAGp* or CCCCACCCCGp*, as substrates
The results presented indicate that E. coli cyclase can efficiently use as substrates 3Ј-phosphate-terminated RNA molecules of different sequence and base composition and that ribonucleoside 3Ј-monophosphates and 5Ј,3Ј-bisphosphates do not act as substrates
Summary
Known enzymes, among them many secretory degradative nucleases such as RNases A or T1, cyclic phosphates are formed as intermediates that are subsequently opened into 3Ј-phosphomonoesters [1, 2]. The only known cellular RNA ligase in eubacteria, which joins RNA ends via the 2Ј,5Ј-phosphodiester, requires 2Ј,3Јcyclic ends for ligation [32, 33] Another finding uncovering a potential role for the 2Ј,3Ј-cyclic phosphate in RNA metabolism was the demonstration that the spliceosomal U6 snRNA in many organisms has a cyclic 2Ј,3Ј-phosphodiester at the terminus. In light of the importance of cyclic termini in RNA metabolism, it was interesting to discover that endonucleolytic cleavage is not the only way to generate RNA molecules bearing 2Ј,3Ј-cyclic phosphates Such molecules can be produced by the action of the RNA 3Ј-terminal phosphate cyclase, an enzyme that catalyzes ATP-dependent conversion of a 3Ј-phosphate at the end of RNA to the 2Ј,3Ј-cyclic phosphodiester [11]. Conservation of the cyclase among eukaryotic and prokaryotic organisms suggests that the enzyme performs an important function in RNA metabolism
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
