A method of inhibiting the expression of particular genes by using external guide sequences (EGSs) has been improved in its rapidity and specificity. Random EGSs that have 14-nt random sequences are used in the selection procedure for an EGS that attacks the mRNA for a gene in a particular location. A mixture of the random EGSs, the particular target RNA, and RNase P is used in the diagnostic procedure, which, after completion, is analyzed in a gel with suitable control lanes. Within a few hours, the procedure is complete. The action of EGSs designed by an older method is compared with EGSs designed by the random EGS method on mRNAs from two bacterial pathogens.S election of accessible sites in target RNAs is critical for efficient mRNA inactivation strategies. Many of the target sequences in cellular RNAs are inaccessible because of the secondary or tertiary structures of the RNA or the binding of proteins to the target RNA in vivo. Several approaches for the mapping of accessible sites in target RNAs have been reported. These approaches range from analyses in silico to cleavage by complex ribozyme constructs (e.g., 1-6), but they are timeconsuming and are not reliable in terms of their efficacy.In Gram-negative bacteria, down-regulation of gene expression at the RNA level has been achieved by directing external guide sequences (EGSs) to pair with complementary regions in mRNAs from individual genes. The EGS will hydrogen-bond to the target RNA and generate an RNA-RNA stem structure mimicking the natural precursor tRNA (ptRNA) cleavage site (7-9). The target RNA in the complex is cleaved by RNase P (10-14).EGSs have been designed to successfully alter several bacterial phenotypes (11)(12)(13)(14). In mammalian cells, RNase P RNA has been specifically directed to destroy tumor-specific fusion genes created as a result of chromosome abnormalities (15) and to inhibit viral gene expression and growth in cell cultures (16,17).Here we describe a rapid method to map directly accessible and cleavable sites in target RNA by the Escherichia coli RNase P holoenzyme and a random EGS (rEGS) library. ResultsDesign of the rEGSe and rEGSx Libraries. The rEGSe and rEGSx libraries were constructed by using a partially randomized oligonucleotide as a template for PCR (Fig. 1). This oligo contains the T7 promoter sequence upstream of the random 14-nt sequence (N 14 ). The length of the randomized region could be varied, but 14 nts were selected because 13-to 16-nt EGSs were shown to work well in previous studies in bacteria (10)(11)(12)(13)(14). In addition, the oligonucleotide contains a cytosine that will base pair to a guanosine in the target RNA sequence and a 3Ј-ACCA sequence. A guanosine is the preferred nucleotide immediately 3Ј of the natural ptRNA cleavage site, and a 3Ј-ACCA sequence in ptRNA is important for cleavage in vivo by E. coli RNase P. The only difference between the rEGSe and rEGSx libraries is that the former contains all of the nucleotides after the BstNI site (Fig. 1), whereas the latter was digested by Bst...

The high-level expression plasmid for streptokinase, pSK100, has been constructed. It contains a tac promoter, an ompA signal sequence, a streptokinase structural gene(skc) and a rrnBT1T2 transcription terminator. E. coli JM109 carrying pSK100 produced about 5,000IU of streptokinase per 1 ml of LB-ampicillin media. About 95% of the expressed streptokinase was secreted into the periplasmic and extracellular fractions. The recombinant streptokinase in high yield and purity may be a potential alternative source for the therapeutic agent.

OLE (ornate, large, and extremophilic) RNA is a noncoding RNA that is found in several extremophilic bacteria, including Bacillus halodurans. The function of OLE RNA has not been clarified. In this study, we found that RNase P cleaves OLE RNA and that the cleavage leads to a small reduction of expression of a downstream gene determined by analyses in vitro and in vivo. Under RNase P-deficient conditions, the amount of OLE RNA increased. Our results imply that RNase P could play a role in the regulation of gene expression in relation to conserved RNA motifs like OLE RNA as well as in riboswitches and operons. Bacillus haloduransO LE (ornate, large, and extremophilic) RNA is a highly conserved RNA motif that is found in several extremophilic Gram-positive eubacteria (1). It was identified with bioinformatic approaches for finding additional riboswitches. However, OLE RNA has different biological characteristics from those of riboswitches and is believed to be a different type of RNA element that is involved in gene regulation (1). In Bacillus halodurans, an extremophilic bacterium widely used for industrial purposes, OLE RNA is located between the BH2780 and BH2781 genes (Fig. 1). Although the functions of OLE and BH2780 RNAs have yet to be understood, OLE RNA can form a complex in vitro with the BH2780 protein, which is a possible transmembrane protein, suggesting that OLE RNA might play a role in a fundamental cellular process as a ribonucleoprotein complex (R. Breaker, personal communication). It is also unknown whether OLE RNA regulates the expression of downstream genes, and it is not apparent that the transcript of OLE RNA extends through the beginning of the BH2780 gene.RNase P is a well known endoribonuclease that is responsible for the 5Ј maturation of tRNA and several RNA molecules, including 4.5S RNA and operons in eubacteria (2). Besides these substrates, RNase P cleaves transient structures of some riboswitches, such as coenzyme B12 riboswitch in Escherichia coli (3,4). Here, we show that RNase P cleaves OLE RNA and that this cleavage can affect the expression of a downstream gene in vivo. This kind of RNase P function might be more general than is anticipated in bacterial species. ResultsRiboswitches are RNA elements regulating gene expression downstream of these elements by responding to metabolites, which are produced by the downstream gene product (4). In a previous report, we showed that cleavages by RNase P lead to the reduction of downstream genes in an operon by using lacZ as a reporter gene (3).␤-Galactosidase Activity in Wild-Type and Mutant Strains of RNase P in E. coli. The effects of the cleavages by RNase P on OLE RNA in vivo were investigated by using a model system with a lacZ reporter gene (Fig. 2). The plasmids pOLE-lacZ and pBADlacZ, a control plasmid, were introduced into NHY312 (wildtype) and NHY322 (RNase P ts ) strains. A wild-type strain, MG1693, and the mutant strain of RNase E, SK5665, also were transformed to identify the effects of one of the major endoribonucleases in E. c...

External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

The artificial inhibition of expression of genes in Saccharomyces cerevisiae is not a widespread, useful phenomenon. The external guide sequence (EGS) technology, which is well-proven in bacteria and mammalian cells in tissue culture and in mice, can also be utilized in yeast. The TOP2 and SRG1 genes can be inhibited by~30% with EGSs in vivo. Results in vitro also show convenient cleavage of the relevant transcripts by RNase P and appropriate EGSs. The feasible constructs shown to date have an EGS covalently linked to M1 RNA, the RNA subunit of RNase P from Escherichia coli. Greater efficiency in cleavage of transcripts can be fashioned using more than one EGS targeted to different sites in a transcript and stronger promoters controlling the EGS constructs.