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The 5' region of the rRNA operon, rrnA, of M. capricolum was cloned. Sequence analysis revealed two tRNA genes, tRNA(leu) and tRNA(lys), upstream to the promoter of the rRNA operon. The in vivo transcription start sites of the rRNA operon and of the tRNA genes were mapped. The same promoters used by M. capricolum RNA polymerase are also recognized by E. coli RNA polymerase both in vivo and in vitro. We find that high levels of ppGpp in E. coli, resulting from amino acid starvation or from spoT mutation, activate rather than repress the transcription of the mycoplasma rrnA operon.

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DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 105 CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.

RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene. The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132. It was concluded that transcription of the rRNA operon of M. pneumoniae is initiated by a single promoter. The nucleotide sequence of the region is presented.The study of the molecular biology of mycoplasmas (class Mollicutes) has revealed them to possess several unique and interesting properties (18). While the group is very diverse, the mycoplasmas share several basic properties, including the lack of a cell wall, the smallest genome among selfreplicating procaryotes (500 to 1,000 megadaltons in size), and an unusually low G+C content in their DNA, usually ranging between 24 and 35 mol% G+C, with Mycoplasma pneumoniae and some anaerobic mycoplasmas being exceptions in having about 40 mol% G+C (19). All the Mollicutes species tested so far have also been found to carry only one or two rRNA operons, whereas other eubacteria usually have a multiplicity of rRNA operons, often more than five (18).The synthesis of rRNA is a very essential process in the growth of any organism. It is not surprising, therefore, that genes for rRNA have been highly conserved among procaryotes (28). Transcription of rRNA operons in Escherichia coli (4) as well as in Mycoplasma capricolum (7) is under negative stringent control. Although much has been learned about this mechanism in E. coli and in several other eubacteria (4), there are many aspects of this complex control system that are still unclear. The study of the structure and function of elements controlling the expression of rRNA operons in mycoplasmas has a special appeal, as the small copy number of these operons in mycoplasmas is expected to provide a simpler system for study than that provided by bacteria having a multiplicity of rRNA operons. In addition, the basic structural sim...