No abstract

The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptidephosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.drug resistance ͉ Gram-positive and Gram-negative bacteria ͉ peptide-phosphorodiamidate morpholino oligonucleotide ͉ RNase P T he utility of bacterial transformation for therapeutic purposes has been limited by the number of species that will undergo transformation and the frequency with which that event happens. To accommodate new therapies that involve small nucleic acids, a means has to be developed to enable bacterial species to take up these nucleic acids with relative ease. The covalent linkage of arginine-rich peptides to the ends of chemically-modified RNAs facilitates the uptake of the RNA analog (1, 2) and other similar molecules (4,5). This methodology in combination with an effective means of inactivating gene expression has to be developed to make it useful for therapeutic agents. There are other processes that function in bacteria to inhibit gene expression (3, 6), but the external guide sequence (EGS) technology (7,8) that is mediated by RNase P cleavage of the target RNA seems optimal in this regard.RNAi and siRNA (ref. 9 and references therein) are not useful tools for the transformation of bacterial species because these RNAs rely on an intracellular complex, the Dicer complex (9) in particular, to release ssRNA that will base-pair with the target mRNA. EGS technology, which is just as effective as siRNA in mammalian cells in tissue culture (10), is very effective in Escherichia coli (11,12) and Salmonella typhimurium (13). Bacterial cells can be altered from drug resistance to drug sensitivity with the methods generally described here (11), and a similar method has also been reported (14). Essential genes can also be inactivated in terms of their expression. The EGS method will allow 3-bp mismatche...

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRS amino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of E. coli PheRS.

Initiation factor 1 (IF1) is an essential protein in Escherichia coli involved in the initiation step of protein synthesis. The protein level of IF1 increases when E. coli cells are subjected to cold shock, however, it remains unclear as to how this increase occurs. The infA gene encoding IF1 contains two promoters, the distal P1 and the proximal P2 promoter. In this study, we found that infA mRNA was greatly increased, and that this increase resulted from transcriptional activation of P1, not P2, during cold shock although stability of transcripts from both promoters concomitantly increased.

M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5 leader sequence of precursor tRNAs. It is generated by the removal of 36 nucleotides from the 3 end of the primary rnpB transcript (pM1 RNA), but the biological significance of this reaction in bacterial metabolism remains obscure. In this study, we constructed and analyzed bacterial strains carrying mutations in the rne-dependent site of their rnpB genes, showing that the 3 processing of M1 RNA is essential for cell viability. Furthermore, we demonstrate that pM1 RNA can undergo not only 3 processing but also poly(A)-dependent degradation. Therefore, our results suggest that the 3 processing of M1 RNA provides a functional mechanism for the protection of its primary transcript against degradation.RNase P was characterized initially as an RNA-processing enzyme that removes the 5Ј leader sequence of precursor tRNAs (1). In addition to precursor tRNAs, non-tRNA substrates of RNase P, such as the 4.5 S RNA, tmRNA precursors, and some polycistronic mRNAs, are also found in Escherichia coli (2-5). RNase P enzymes from diverse organisms have been shown to contain both essential RNA and protein components (6, 7). The E. coli holoenzyme consists of two subunits, an RNA subunit (M1 RNA, 377 nucleotides), and a small basic protein (C5 protein, 119 amino acids). M1 RNA, as a naturally occurring ribozyme, carries out the catalytic reaction in the absence of C5 protein in vitro (8), although both components are essential for the activity of RNase P in vivo (9).M1 RNA is derived from the major primary transcript from position ϩ1 of the rnpB gene (10), which terminates at position ϩ413 (11). This precursor transcript, pM1 RNA, subsequently undergoes a 3Ј processing reaction to generate a mature M1 RNA species of 377 nucleotides. This reaction is initiated with a cleavage by RNase E at an rne-dependent site to produce processing intermediates with 3Ј ends of ϩ378 and ϩ379 (12-14), which are subsequently trimmed to ϩ377 by exoribonucleases (15). Although the M1 RNA processing has been a well studied process (13-16), the biological significance of this process remains unclear. One possible function could be the generation of a functional RNA molecule, but as both pM1 RNA and M1 RNA have comparable RNase P activity (17) and M1 RNA retains its catalytic activity with the additional seven nucleotides at its 3Ј end (18), this would seem unlikely. Hence, the question of why this processing reaction needs to occur in the cell is an intriguing one.In this study, we constructed mutant strains containing alterations in their rne-dependent sites at the chromosomal rnpB locus and analyzed the resulting phenotypes. The mutant cells showed growth defects, which correlated with their M1 RNA levels, and the analysis of M1 RNA metabolism in both wild-type and mutant cells indicated that pM1 RNA undergoes not only 3Ј processing but also poly(A)-dependent degradation. These results suggest that the processing of M1 RNA is required for the prote...