The activity of ribonuclease P on precursor tRNA substrates from Escherichia coli can be abolished by pretreatment of this enzyme with micrococcal nuclease or pancreatic ribonuclease A, as well as by proteases and by thermal denaturation. Highly purified RNase P exhibits one prominent RNA and one prominent polypeptide com nent when examined in polyacrylamide gels containing sodum dodecyl sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is characteristic of a protein-RNA complex. The activity of RNase P is inhibited by various RNA molecules. The presence of a discrete RNA component in RNase P appears to be essential for enzymatic function. A model is described for enzyme-substrate recognition in which this RNA component plays an important role.Ribonuclease P (1-3) is necessary for the biosynthesis of the 5' termini of tRNA molecules in Escherichia coli. This enzyme recognizes some aspect of the structural conformation rather than nucleotide sequence at is cleavage sites in tRNA precursor molecules (4-6). Exactly how this recognition occurs is a matter for speculation (7). In this report we show that treatment of highly purified RNase P with either micrococcal nuclease (MN) or pancreatic ribonuclease A abolishes RNase P activity. Our most highly purified RNase P preparations contain one discrete RNA species and one discrete protein species, and several kinds of RNA can inhibit the enzymatic activity. We conclude that the interaction of RNase P with its substrates depends on the presence of RNA in the enzyme complex. METHODS Preparation of RNase P. What follows is an abbreviated description of RNase P purification schemes. Complete details will be published elsewhere. Crude extracts (S30) of E. coil MRE 600 were prepared by grinding of frozen cells with alumina as described (2). Two purification schemes were used, starting with S30.Scheme I. S30 made from 200 g of cells was diluted 1:5 with buffer A [50 mM Tris-HCl, pH 7.5/60 mM NH4Cl/10 mM Mg(OAc)2/6 mM 2-mercaptoethanol] and loaded onto a DEAE-Sephadex A-50 column (12 X 25 cm) equilibrated with buffer A. Bed volume of the column was about 2.5 liters. The column was washed successively with 3 liters of buffer A containing 0.2 M NH4Cl, 0.3 M NH4Cl, and 0.4 M NH4Cl in successive washes. The final wash was with buffer A containing 0.5 M NH4Cl (now called buffer B) and the activity was eluted after about 1 liter of the last wash buffer had flowed through the column. The pooled active fractions wer-precipitated with 0.6 g of (NH4)2SO4 per ml; the precipitate was resuspended and dialyzed against buffer B to give a final volume of about 15 ml. This material was applied to a Sepharose 4B column (2.5 X 90 cm) equilibrated with buffer B and eluted with 300 ml of the same buffer. The RNase P eluted after the rRNA peak and before the 4S RNA peak. The active fractions were pooled and concentrated as described above and then applied to a Sephadex The active RNase P in the salt wash was loaded onto a DEAESephadex column and purified as described (2). RNase P (...

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.

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The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.