Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate.

Transcription terminators recognized by several RNA polymerases include a DNA segment encoding uridine-rich RNA and, for bacterial RNA polymerase, a hairpin loop located immediately upstream. Here, mutationally altered Escherichia coli RNA polymerase enzymes that have different termination efficiencies were used to show that the extent of transcription through the uridine-rich encoding segment is controlled by the substrate concentration of nucleoside triphosphate. This result implies that the rate of elongation determines the probability of transcript release. Moreover, the position of release sites suggests an important spatial relation between the RNA hairpin and the boundary of the terminator.

Kimura disease: review of the literature.

Kimura disease (KD) is a rare, chronic inflammatory disease of unknown cause and is characterized by painless s.c. swellings and lymphadenopathy commonly affecting the head and neck region. Much therapeutics has been used to treat KD, but is not satisfactory because of frequent relapse. Imatinib has been reported previously to be useful for treatment of hypereosinophilic syndrome and may work by selectively blocking protein-tyrosine kinases, such as platelet-derived growth factor receptor, and c-Kit. We carried out immunohistochemical examination of platelet-derived growth factor receptor-alpha and c-Kit in tissues from patients with KD. The results were positive and suggested that Imatinib might be an effective drug for the treatment of the disease. We have also briefly reviewed the epidemiology, aetiology, clinical manifestations, laboratory and pathological examinations, differential diagnoses, treatment and prognosis of KD in this manuscript.

An Escherichia coli RNA polymerase defective in transcription due to its overproduction of abortive initiation products.

One of the potential regulatory steps in procaryotic transcription is promoter clearance, a transition step in transcription initiation at which an RNA polymerase (RNAP) switches from the initial transcribing stage to the elongation stage. The biological significance of promoter clearance and the role of RNAP in this process are not understood. One approach to address these questions is to study mutant RNAPs that have altered promoter clearance. Because the antibiotic rifampicin inhibits transcription by preventing an initial transcribing complex from entering the elongation mode, mutant RNAPs which confer rifampicin (Rifr) are likely to be altered in promoter clearance. To test this hypothesis, we studied the effects of Rifr RNAPs on the pyrBI promoter, which is subject to control of promoter clearance in response to the availability of UTP. Two Rifr alleles that carry a different altered amino acid residue at position 529 of the beta subunit appeared to be defective in transcription from the pyrBI promoter in vivo. Biochemical analysis of one of these mutant RNAPs, RpoB3401 with a R529C change in the beta subunit, showed that it overproduces aborted initiation products from the pyrBI promoter and thus is defective in promoter clearance leading to reduced productive initiation. The severity of overproducing the aborted initiation products is an inverse function of the UTP concentration indicating that RpoB3401 has reduced affinity for UTP and thus is subject to a high Km barrier during promoter clearance. The defect of RpoB3401 in abortive initiation in vitro could account fully for its reduced initiation activity in vivo demonstrating the biological significance of abortive synthesis in transcription initiation. Our results indicate that at least part of the "rif region" is important for the process of abortive initiation and that promoter clearance can be regulated in part by modulating the Km of RNAP for nucleotides during initiation. The mutant enzyme is not altered in stuttering RNA synthesis at the pyrBI promoter, previously observed with wild-type RNAP. Our results also show that the mechanisms underlying the two non-productive initiation events (abortive synthesis and stuttering synthesis) at the pyrBI promoter are distinct.

Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance.

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.

A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022.

Antitermination of early transcription in phage HK022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. It does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. To identify host proteins involved in antitermination, we isolated 14 Escherichia coli mutants that are specifically blocked in HK022 growth. The mutations are located in the rpoC gene, which encodes the beta' subunit of RNA polymerase. Each mutation alters one of three amino acid residues located within a cluster of four completely conserved cysteine residues that are believed to bind zinc. We examined the effect of one mutation on HK022 antitermination in vivo. rpoCY75N greatly reduced readthrough of a strong rho-independent transcription terminator placed downstream of the HK022 PL promoter and nutL analog, but did not decrease promoter activity. Purified enzyme had a similar effect on PL-directed transcription in vitro: wild-type but not mutant polymerase read through a strong rho-independent terminator located immediately downstream of the nutL analog with high efficiency. We suggest that interaction of the putative zinc-binding domain of the RNA polymerase beta' subunit with the HK022 antitermination sites suppresses transcription termination, and that this interaction can occur in the absence of other proteins.

Genetic evidence for the interaction between cluster I and cluster III rifampicin resistant mutations.

Rifampicin-resistant (Rifr) mutations of Escherichia coli map to the central portion of the rpoB gene, which encodes the beta subunit of RNA polymerase. These mutations are located in three distinct clusters, designated I, II and III. Three intragenic suppressors of the cluster III Rifr mutation, rpoB3406(RH687), restore the ability of the mutant strain to grow at low and high temperatures and map to a single locus in cluster I. These suppressors are identical to two previously characterized Rifr alleles, rpoB3401(RC529) and rpoB3402(RS529). None of the other 14 previously identified Rifr mutations that we have characterized confers this phenotype. We suggest that this allele-specific suppression results from interaction between Cluster I and Cluster III of the beta subunit.

The mechanism of early transcription termination by Rho026.

We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene. Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt'. The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified. We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE. The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation. We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants. NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG. However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE. These results suggest that NusG aids in the 3' end formation process. The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.

Characterization of the termination phenotypes of rifampicin-resistant mutants.

Rifampicin-resistant (Rifr) mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have examined the effect of each of the 17 sequenced Rifr mutations in our collection on transcription termination. The effect of each Rifr mutation was measured at three types of terminators: simple terminators requiring only RNA polymerase to terminate in vitro, and complex terminators requiring either Rho or Tau for in-vitro termination. Almost every Rifr allele examined (14/17) affected readthrough at one or more of these terminators. We found that mutations with similar termination phenotypes were clustered suggesting functional specialization within the region of rpoB defined by the Rifr mutations. The interaction of the Rifr mutations with the defective rho15 allele was also investigated. Only two Rifr mutations suppress the termination defect of rho15 strains. We discuss models to explain how this region of the beta polypeptide might be involved in the process of transcription termination.

Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli.

Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E. coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes or stable RNA. We have found that Rifr mutations affect both lambda N-mediated antitermination and the cellular antitermination system involved in synthesis of stable RNA. Because NusA is involved in antitermination and termination, we also investigated the interaction of NusA and RNA polymerase by determining whether Rifr mutations alter NusA-dependent termination or antitermination in cells with defective nusA alleles. We have shown that Rifr mutations can either enhance or suppress the phenotypes of defective nusA alleles. Most Rifr mutations alter the temperature range over which the nusA1 allele supports lambda N-mediated antitermination. In addition, a number of Rifr alleles restore termination to the nusA10(Cs) and the nusA11(Ts) mutants defective in this process. Our results indicate that the region of the rpoB gene defined by the Rifr mutations is involved in the antitermination process and affects the activity of the NusA protein directly or indirectly.

Validated HPLC Method for the Determination of Paclitaxel-related Substances in an Intravenous Emulsion Loaded with a Paclitaxel-Cholesterol Complex.

A high-performance liquid chromatography method was developed for the determination of related substances in an intravenous emulsion loaded with a paclitaxel-cholesterol complex. The separation was achieved using Agilent Eclipse XDB-C18 column (150×4.6 mm, 3.5 µm), which was kept at 40°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1.2 ml/min. The ultraviolet detection wavelength was set at 227 nm. The preparation of the sample solution began with the addition of anhydrous sodium sulphate to break the emulsion. Then, methanol and ethyl ether were added to pick up the drug and remove the accessories of the emulsion by extraction and centrifugation. Finally, paclitaxel was enriched by a nitrogen blow method and resolved with a mixture of methanol:glacial acetic acid (200:1). The method was proven to be selective, sensitive, robust, linear, repeatable, accurate and suitable for the determination of paclitaxel-related substances in the emulsion formulations, and the major degradation products in the potential pharmaceutical product were 7-epipaclitaxel and 10-deacetylpaclitaxel.

Phenolic glycosides from leaves of Hopiciopsis lobata.

A new phenolic glycoside, 6'-[(E)-2''-hydroxymethyl, 2''-butenoyl] arbutin (1), and two known phenolic glycosides, 6'-[(E)-4''-hydroxycinnamoyl] arbutin (2) and 6'-[(E)-3'',4''-dihydroxycinnamoyl] arbutin (3), were isolated from the leaves of Heliciopsis lobata (Merr.) Sleum. Their structures were elucidated by various spectroscopic methods including 2D NMR spectroscopy.

Slippage synthesis at the galP2 promoter of Escherichia coli and its regulation by UTP concentration and cAMP.cAMP receptor protein.

An intriguing mechanism in regulating transcription initiation from the gal operon in Escherichia coli is described. Initiation from galP2, one of the two promoters of the E. coli galactose operon, is shown to be subject to promoter clearance control in responding to changes in UTP concentration. In vitro, RNA polymerase (RNAP) makes a large amount of nonproductive "stuttering" initiation products at the galP2 promoter at high concentrations of UTP and less of the stuttered products at low concentrations of UTP. Conversely, RNAP makes more productive initiation products at low UTP concentration than at high UTP concentration. The transcription factor cAMP.CRP complex which normally inhibits transcription from galP2 also represses the stuttering synthesis from galP2. When galactose is used as a sole carbon source and the internal UTP pools are adjusted externally, a cya mutant (in which galP2 is mainly responsible for the expression of the gal operon and galP1 activity is minimal) has a slower growth rate and lower expression of the gal operon at high UTP pools than at low UTP pools. Such an apparent correlation between the in vitro and in vivo results allows one to speculate that changes in UTP concentration can modulate the expression of the gal operon. The implication of a gal promoter being controlled by UTP is discussed.

A mutant RNA polymerase reveals a kinetic mechanisms for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance.

During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP. This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2. RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP. Thus, a direct linkage between promoter clearance and productive transcription is demonstrated. The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied. Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2. Because RpoB3449 demonstrates "wild-type" stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context. Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation. A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.

RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation.

The rpoB8 allele of Escherichia coli maps to the beta-subunit of RNA polymerase and confers rifampicin resistance as well as increased termination at both intrinsic and rho-dependent terminators in vivo. This phenotype suggests that the mutant is defective in an enzymatic property of RNA polymerase important for all termination events. We analyzed the in vitro transcription properties of this enzyme to determine the nature of the defect. As compared with the wild-type enzyme, RpoB8 exhibits enhanced pausing and a significant reduction in rate of elongation on natural templates. In addition, RpoB8 RNA polymerase has a 3-5-fold higher Km for purine nucleotides during elongation on synthetic templates. In contrast, both the mutant and wild-type enzyme have the same initiation Km for ATP. Kinetic analysis indicates that RpoB8 is likely to be defective in nucleotide binding during elongation, suggesting that the mutational alteration affects the binding site. We show that our data are consistent with the idea that the altered Km underlies the altered pausing and elongation properties of the enzyme, and we discuss the implication of these results for the termination proficiency of the mutant strain.

RNA polymerase beta mutations have reduced sigma70 synthesis leading to a hyper-temperature-sensitive phenotype of a sigma70 mutant.

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli.

In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but "turned off" under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like "stringent" RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

RapA, a novel RNA polymerase-associated protein, is a bacterial homolog of SWI2/SNF2.

We have identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase named RapA. Almost all of this 110-kDa protein in the cell copurifies with RNAP holoenzyme as a 1:1 complex. Purified to homogeneity, RapA also forms a stable complex with RNAP, as if it were a subunit of RNAP. The ATPase activity of RapA is stimulated by binding to RNAP, and thus, RapA and RNAP interact physically as well as functionally. Interestingly, RapA is a homolog of the SWI/SNF family of eukaryotic proteins whose members are involved in transcription activation, nucleosome remodeling, and DNA repair.

Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli.

We used our collection of 17 sequenced rifampin resistance alleles in rpoB to perform a systematic analysis of the phenotypes historically reported with this class of mutants, including growth phenotype, ability to support the growth of different bacteriophages, ability to maintain the F' episome, interaction with mutant alleles at other loci, sensitivity to uracil, inhibition by 5-fluorouridine, and dominance. We found that mutational changes leading to the same phenotype were often located together and that certain phenotypes were associated with one another.

Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants.

We have mapped three Escherichia coli RNA polymerase mutations selected by Guarente (1979) to suppress the termination defects of rho201. We find that two of the mutations are located in the 3' half of the rpoB gene encoding the beta subunit. The third mutation is in the rpoC gene, encoding the beta' subunit. All three RNA polymerase mutations affect termination efficiency, even in rho+ strains, suggesting that the C-terminal end of the beta as well as the beta' subunit participates in termination. In addition we find that all three rpoBC alleles inhibit lambda N-mediated antitermination at 30 degrees C in a strain containing the nusA1 allele. It may be significant that the three other RNA polymerase mutations known to revert the termination defect of mutant rho alleles also affect N-mediated antitermination in nusA1 strains. The correlation of these two phenotypes suggests that both phenotypes may arise from the same functional defect in RNA polymerase.