Effects of base changes at the transcription start site on stringent control of rnpB in Escherichia coli.

The GC-rich discriminator sequence between the -10 region and the transcription start site of the rnpB promoter is responsible for stringent control of M1 RNA synthesis. The rnpB promoter also contains a G nucleotide at the previously identified transcription start site. In this study, we examined by mutagenesis of G to A whether this +1G nucleotide is involved in the stringent response.

Germ cell-specific gene 1 targets testis-specific poly(A) polymerase to the endoplasmic reticulum through protein-protein interactions.

Testis-specific poly(A) polymerase (TPAP) is a cytoplasmic poly(A) polymerase that is highly expressed in round spermatids. We identified germ cell-specific gene 1 (GSG1) as a TPAP interaction partner protein using yeast two-hybrid and coimmunoprecipitation assays. Subcellular fractionation analysis showed that GSG1 is exclusively localized in the endoplasmic reticulum (ER) of mouse testis where TPAP is also present.

Dual function of RNase E for control of M1 RNA biosynthesis in Escherichia coli.

M1 RNA, the gene product of rnpB, is the catalytic subunit of RNase P in Escherichia coli. M1 RNA is transcribed from a proximal promoter as pM1 RNA, a precursor M1 RNA, and then is processed at its 3' end by RNase E. In addition to pM1 RNA, large rnpB-containing transcripts are produced from unknown upstream promoters.

ERK is a novel regulatory kinase for poly(A) polymerase.

Poly(A) polymerase (PAP), which adds poly(A) tails to the 3' end of mRNA, can be phosphorylated at several sites in the C-terminal domain. Phosphorylation often mediates regulation by extracellular stimuli, suggesting PAP may be regulated by such stimuli. In this study, we found that phosphorylation of PAP was increased upon growth stimulation and that the mitogen-activated protein kinase ERK was responsible for the increase in phosphorylation.

Tin-free radical alkylation of ketones via N-silyloxy enamines.

The radical alkylation of ketones is achieved by their conversion into corresponding N-silyloxy enamines, followed by a radical reaction with alkyl halides bearing electron-withdrawing groups.

Coupled expression of MhpE aldolase and MhpF dehydrogenase in Escherichia coli.

MhpE (4-hydroxy-2-ketovalerate aldolase) and MhpF [acetaldehyde dehydrogenase (acylating)] are responsible for the last two reactions in the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway in Escherichia coli, which is homologous to the meta-cleavage pathway in Pseudomonas species. Here, we report that the MhpE aldolase is associated with the MhpF dehydrogenase and that MhpF is indispensable for the folding of MhpE. Moreover, our results suggest that the mhpF and mhpE genes are translationally coupled through a reinitiation mechanism.

Differential promoter usage of infA in response to cold shock in Escherichia coli.

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.

Processing of m1 RNA at the 3' end protects its primary transcript from degradation.

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.

Effects of FIS protein on rnpB transcription in Escherichia coli.

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs.

Kinetic analysis of precursor M1 RNA molecules for exploring substrate specificity of the N-terminal catalytic half of RNase E.

To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E). NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates. The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency.

Regulation of 6S RNA biogenesis by switching utilization of both sigma factors and endoribonucleases.

In Escherichia coli, 6S RNA functions as a modulator of RNA polymerase sigma70-holoenzyme activity, but its biosynthetic pathway remains uncharacterized. In this study, to further understand the regulatory circuit of 6S RNA biosynthesis for the modulation of Esigma70 activity, we have characterized the biogenesis of 6S RNA. We reveal that there are two different precursors, a long and a short molecule, which are transcribed from the distal P2 and proximal P1 promoter, respectively.

Glycosylation of onconase increases its conformational stability and toxicity for cancer cells.

Onconase (ONC) is currently in Phase III clinical trials as a cancer chemotherapeutic agent. Despite the finding that ONC contains an N-linked glycosylation site (-N69-V70-T71-), only the non-glycosylated form of the protein has been identified to date. We employed the Pichia pastoris expression system to produce recombinant glycosylated ONC (gONC) protein.

Alteration of stringent response of the Escherichia coli rnpB promoter by mutations in the -35 region.

It is well known that the GC-rich discriminator region between the -10 region and the transcription start site is important for the stringent control of the transcription. However, the discriminator activity is influenced by flanking regions, in particular in conjunction with the promoter -35 and -10 sequences. In this study, we changed the sequence in the -35 region of the rnpB P-1 promoter to see how such changes affect the stringent control.

Role of the sequence of the rne-dependent site in 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P.

The 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps. The precursor of M1 RNA has an rne-dependent site downstream of the processing site, whose sequence variation affects the processing efficiency. In this study, we showed that the sequence itself of the rne-dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites.

Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect on Candida albicans.

Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C.

Testis-specific expression of an intronless gene encoding a human poly(A) polymerase.

A mouse intronless gene, encoding a testis-specific poly(A) polymerase (mPAPT), was previously identified. mPAPT may play a role as a putative enzyme that is responsible for polyadenylation regulation during mouse spermatogenesis. In order to understand how PAPT genes are conserved in mammals, we isolated a human cDNA homolog encoding a human PAPT (hPAPT), which was specifically expressed in the testis.