RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

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Mycobacterium tuberculosis is a natural mutant in oxyR, a close homolog of the central regulator of peroxide stress response in enteric bacteria. Inactivation of oxyR is specific for M. tuberculosis and other members of the M. tuberculosis complex. This phenomenon appears as a paradox due to the ability of this organism to parasitize host macrophages, in which the ingested organisms are likely to be exposed to reactive oxygen intermediates. However, the surprising finding that M. tuberculosis has multiple deletions, nonsense and frameshift mutations in oxyR may help explain the exceptionally high sensitivity of M. tuberculosis to the potent antituberculosis agent isoniazid. One of the genes affected by oxyR lesions, ahpC (encoding an alkylhydroperoxide reductase) may determine the intrinsic sensitivity of mycobacteria to isoniazid.

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and likeMycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate geneahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to theoxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region ofahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katGexpression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream ofkatG in M. marinum. The furA-katGlinkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC andkatG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.