Journal of Molecular Biology
CommunicationAnalysis of Putative RNase P RNA from Orthopoxviruses
Section snippets
Cloning and transcription in vitro
A sequence of camelpox virus was found to be similar in structure and conserved sequences to RNase P RNA3 and could be folded into a putative RNase P RNA (Figure 1). A DNA fragment that encoded this putative camelpox virus RNase P RNA (CPVRPR) was synthesized and inserted into pBT7 vector4 to generate the pBT7D-CPVRPR plasmid. The CPVRPR gene was under the control of a T7 promoter and the insert sequence was verified experimentally. The first nucleotide in this sequence was chosen to be the
RNase P activity of camelpox virus RNase P RNA
CPVRPR in buffers that contained 100 mM MgCl2 showed no obvious cleavage activity on Escherichia coli precursor to tRNATyr (pTyr). An attempt was made to reconstitute CPVRPR in vitro with E. coli RNase P protein subunit C5.5 No cleavage activity was detected during a 60 min incubation. Some other precursor tRNAs (E. coli precursor to tRNAPhe (pPhe) and human precursor to tRNAfMet (pfMet)) were also used in an assay in vitro but no cleavage activity was observed from CPVRPR (data not shown).
RNase P activity of camelpox virus RNase P RNA in a camel cell extract
Camel fibroblast cell S16 extract6, 7, 8 was prepared for reconstitution of RNase P in vitro. Incubation of CPVRPR and camel fibroblast cell S16 extract was carried out through the use of pPhe that was used as substrate for RNase P in buffer H (C. Guerrier-Takada, unpublished results). The camel cell extract itself demonstrated RNase P cleavage activity (Figure 2(a), lane 1) and 2 pmol of CPVRPR added to the camel cell extract demonstrated no additional RNase P activity (Figure 2(a), lane 2).
RNase P assay of orthopoxvirus RNase P RNA in infected HeLa cells
With pPhe as a substrate, both HeLa and vaccinia-infected HeLa cells (a gift of Bernard Moss, NIH/NIAID) showed the same RNase P cleavage activity with no CPVRPR added (Figure 3(a), lane 1). The activity of vaccinia RNase P could not to be distinguished from HeLa RNase P enzyme in the vaccinia-infected HeLa cells. Furthermore, the RNase P cleavage activity showed no increase with CPVRPR added (Figure 3(a), lane 2). A considerable reduction (about 20%) of RNase P cleavage activity was found by
Endogenous expression of orthopoxvirus RNase P RNA in vaccinia-infected HeLa cells
Sequence analysis showed that the vaccinia RNase P RNA gene overlaps with an upstream open reading frame (ORF) for a hypothetical protein and a downstream ORF for ATP-dependent DNA ligase (Figure 4(a), top panel).12, 13, 14 The first nucleotide of putative RNase P RNA, taken to be the nucleotide that was identified in the gene search mechanism,3 is a one nucleotide frame shift (+1) removed in relation to the upstream ORF. These aspects of sequence organization were similar in the camelpox virus
Conclusion
We report the cloning and functional analysis of the putative RNase P RNA gene from camelpoxvirus, an orthopox virus. No RNase P activity could be detected in vitro from CPVRPR alone, or after the addition of the RNase P protein subunit from E. coli or a crude extract from camel cells. When using vaccinia virus-infected HeLa cells, vaccinia RNase P activity was not identified, even though the transcription of the exogenous, putative RNase P RNA subunit of vaccinia was confirmed by Northern blot
Acknowledgements
We are grateful to our colleagues, especially Dr Cecilia Guerrier-Takada, for helpful discussions. We also thank Dr Bernard Moss of NIH/NIAID (Bethesda) for generously providing the vaccinia-infected HeLa cells. L. Y. is supported by a Focused Giving grant from the Johnson and Johnson Co. Yale University assisted S. A.
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