RNA polymerase

The sequence of the four large (L) double-stranded RNAs (dsRNAs) associated with Amasya cherry disease (ACD), which has a presumed fungal aetiology, is reported. ACD L dsRNAs 1 (5121 bp) and 2 (5047 bp) potentially encode proteins of 1628 and 1620 aa, respectively, that are 37 % identical and of unknown function. ACD L dsRNAs 3 (4458 bp) and 4 (4303 bp) potentially encode proteins that are 68 % identical and contain the eight motifs conserved in RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with those of members of the family Totiviridae. Both terminal regions share extensive conservation in all four RNAs, suggesting a functional relationship between them. As ACD L dsRNAs 1 and 2 do not encode RdRps, both are probably replicated by those from either ACD L dsRNA 3 or 4. Partial characterization of the equivalent L dsRNAs 3 and 4 associated with cherry chlorotic rusty spot revealed essentially identical sequences.

A Crystallogral structure is described for the Mg2+-BeF3--bound receiver domain of Sinorhizobium meliloti DctD bearing amino acid substitution E121K. Differences between the apo- and ligand-bound active sites are similar to those reported for other receiver domains. However, the off and on states of the DctD receiver domain are characterized by dramatically different dimeric structures, which supports the following hypothesis of signal transduction. In the off state, the receiver domain and coiled-coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation, the receiver domain cannot be phosphorylated or bind Mg2+ and BeF3-. Instead, these modifications stabilize an alternative dimeric conformation that repositions the subunits by approximately 20 A, thus replacing the a4-b5-a5 interface with an a4-b5 interface. Reoriented receiver domains permit the ATPase domain to oligomerize and stimulate open complex formation by the s54 form of RNA po...

Transcription reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated reinitiation during subsequent cycles is compromised.

Rotavirus single-shelled particles have several enzymatic activities that are involved with the synthesis of capped mRNAs both in vivo and in vitro. Because single-shelled particles must be structurally intact to carry out transcription, it has proven to be difficult to identify the protein within such particles that possesses associated RNA polymerase activity. One approach for characterizing the function of the individual proteins within single-shelled particles is to use nucleotide analogs to specifically label those proteins, such as the viral RNA polymerase, that have affinity for nucleotides. In this study, 8-azido-ATP (azido-ATP), a photoreactable nucleotide analog, was used to identify the viral RNA polymerase on the basis of the ability of the analog to inhibit transcription activity associated with rotavirus particles on exposure to UV light. When single-shelled particles were treated with UV light in the presence of [alpha-32P]azido-ATP, the structural protein VP1 became ...

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive sense ssRNA molecule of 8,506 nucleotides. It is predicted to encode a single polyprotein of 314.5 kDa, which is subsequently processed into three coat protein components of 13.7, 17.9 and 13.5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease, and RNA-dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19-47% identity). Evidence of replication in plants was detected by RT-PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. We propose the name Tomato matilda virus (TMaV), and further, propose that the genus Tomavirus (Tomato Matilda virus) be created within the family Iflaviridae. This is the first report of a plant-infecting virus resembling members of the Iflaviridae.

In a previous study, we observed marked synergy between daptomycin and rifampicin against 73% of rifampicin-resistant, vancomycin-resistant Enterococcus faecium (VRE), with approximately 100-fold reductions in rifampicin MICs observed at one-eighth to one-fourth daptomycin MIC. The purpose of this study was to determine whether the synergy between daptomycin and rifampicin could be explained by enhanced entry of rifampicin into the cell or was related to amino acid substitutions in the rifampicin-binding site in the b subunit (rpob) of the RNA polymerase. Methods: We developed a bioassay for rifampicin to measure cell-bound rifampicin levels as well as metabolic inactivation of rifampicin. In addition, we sequenced the rifampicin-binding site in the rpob of VRE strains with and without synergy between daptomycin and rifampicin. Results: Cell-bound rifampicin levels were the same in rifampicin-susceptible VRE as in rifampicinresistant VRE showing daptomycin synergy and were not affected by the presence of daptomycin. In contrast, rifampicin-resistant VRE without daptomycin synergy had undetectable cell-bound rifampicin. Sequencing the rpob rifampicin-binding site revealed that the synergistic strains had the same sequence as rifampicin-susceptible wild-type E. faecium. The daptomycin synergy-resistant strains all had mutations in known rifampicin-binding sites. Conclusions: Daptomycin is able to reverse rifampicin resistance in some strains of VRE, but the mechanism could not be explained by an effect of daptomycin on entry of rifampicin into or transport out of the cell, by inactivation of rifampicin or by mutation involving the rifampicin-binding site.

Although rifamycins have excellent activity against Chlamydophila pneumoniae and Chlamydia trachomatis in vitro, concerns about the possible development of resistance during therapy have discouraged their use for treatment of chlamydial infections. Rifalazil, a new semisynthetic rifamycin with a long half-life, is the most active antimicrobial against C. pneumoniae and C. trachomatis in vitro, indicating its potential for treatment of acute and chronic C. pneumoniae and C. trachomatis infections. We investigated the effect of serial passage of two C. pneumoniae isolates and two serotypes of C. trachomatis in subinhibitory concentrations of rifalazil and rifampin on the development of phenotypic and genotypic resistance. C. trachomatis developed resistance to both antimicrobials within six passages, with higher level resistance to rifampin (128 to 256 μg/ml) and lower level resistance to rifalazil (0.5 to 1 μg/ml). C. pneumoniae TW-183 developed only low-level resistance to rifampin ...

The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P. fluorescens F113 phlF mutant. The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a σ 70 RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene. The repression by PhlF is due to its interaction with an inverted repeated sequence, phO , located downstream ...

Following our recent report showing the potential of naturally occurring aurones (2benzylidenebenzofuran-3(2H)-ones) as anti-hepatitis C virus (HCV) agents, efforts were continued in order to refine the structural requirements for the inhibitory effect on HCV RNA-dependent RNA polymerase (RdRp). In this study, we targeted the Bring moiety of aurones with the aim to improve structural features associated with higher inhibition of the targeted polymerase. In vitro evaluation of the RdRp inhibitory activity of the 37 newly synthesized compounds pointed out that the replacement of the Bring with a N-substituted indole moiety induced the highest inhibitory effect. Of these, compounds 31, 40 and 41 were found to be the most active (IC50 = 2.3-2.4 µM). Docking experiments performed with the most active compounds revealed that the allosteric thumb pocket I of RdRp is the binding pocket for aurone analogues.

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With almost no consensus promoter sequence in prokaryotes, recruitment of RNA polymerase (RNAP) to precise transcriptional start sites (TSSs) has remained an unsolved puzzle. Uncovering the underlying mechanism is critical for understanding the principle of gene regulation. We attempted to search the hidden code in ∼16,500 promoters of 12 prokaryotes representing two kingdoms in their structure and energetics. Twenty-eight fundamental parameters of DNA structure including backbone angles, basepair axis, and interbasepair and intrabasepair parameters were used, and information was extracted from x-ray crystallography data. Three parameters (solvation energy, hydrogen-bond energy, and stacking energy) were selected for creating energetics profiles using in-house programs. DNA of promoter regions was found to be inherently designed to undergo a change in every parameter undertaken for the study, in all prokaryotes. The change starts from some distance upstream of TSSs and continues pas...

The influence of mutations in structural genes of/3 and/3' subunits of RNA polymerase upon the synthesis of these subunits in E. coli cells have been investigated. An amber-mutation ts22 in the/3 subunit gene decreases the intracellular concentration of this subunit and the rate of its synthesis. At the same time the concentration and the rate of/3' subunit synthesis is increased. These suggest the compensatory activation of the RNA polymerase operon that takes place under the conditions of shortage of one of the subunits. Reversions, as well as more effective supression of ts22 amber mutation, achieved by streptomycin addition, substitution of su2 by sul, or by specific mutations, result in a rise of /3 and drop of /3' subunit concentration and synthesis in ts22 mutant. TsX missense-mutation in the /3' subunit gene alters the properties of the enzyme increasing, at the same time, the concentration and the rate of synthesis of both/3 and/3' subunits, particularly at a nonpermissive temperature. This points to an inversely proportional relationship between the rate of synthesis of RNA polymerase subunits and the total intracellular activity of the enzyme. Extra subunits are rapidly degraded in ts22 and tsX mutants. The whole complex of our data and those of others suggest that the regulation of the synthesis of RNA polymerase subunits is accomplished by interaction of a negative and apositive mechanisms of regulation which include not only activators and repressors but the enzyme itself as well.

Multiple interactions with DNA, RNA and transcription factors occur in a transcription cycle. To survey the proximity of some of these factors to the Escherichia coli RNA polymerase surface, we produced a set of nine monoclonal antibodies (mAbs) against the enzyme. These mAbs, located at different places on the surface of the enzyme, were used in a co-immunopurification assay to investigate interference with the binding of NusA, σ70, GreB and HepA to core RNA polymerase. One of these mAbs turned out to be the first antibody inhibitor of the binding of NusA and σ70 ; it did not affect GreB and HepA interactions. Its Abbreviations used : mAb, monoclonal antibody ; Ni-NTA, Ni 2 +-nitrilotriacetate ; SPR, surface plasmon resonance. 1 To whom correspondence should be addressed (e-mail jpIleo!yahoo.com). epitope was located on the βh subunit at the C-terminus of region G. The properties of this mAb reinforce the idea that the mutually exclusive binding of NusA and σ70 to core RNA polymerase is due to, at least partially, overlapping binding sites, rather than allosteric interaction between two distant binding sites. This mAb is also useful to understand the occupancy of σ70, NusA and Gre proteins on core RNA polymerase.

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in Staphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either A or B from S. aureus. Of the seven A-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3, cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the B-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His‐tagged ZntR and purified by Ni2+‐affinity column. His‐tag‐free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 × 10−12 M. The addition of 25 μM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleot...

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner inStaphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either ςA or ςB from S. aureus. Of the seven ςA-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3,cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the ςB-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure. In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation. We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II). Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect. In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo. Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase. Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6. These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al. 1998), provide strong evidence that these factors are important for transcription elongation in vivo.

Oxidized RNA precursors formed in the nucleotide pool may be incorporated into RNA. In this study, the incorporation of 8-hydroxyguanosine 5'-triphosphate (8-OH-GTP, 8-oxo-7,8-dihydroguanosine 5'-triphosphate) into RNA by Escherichia coli RNA polymerase was examined in vitro, using a primer RNA and a template DNA with defined sequences. 8-OH-GTP was incorporated opposite C and A in the template DNA. Surprisingly, 8-OH-GTP was quite efficiently incorporated by the bacterial RNA polymerase, in contrast to the incorporation of the 2'-deoxyribo-counterpart by DNA polymerases, as indicated by the kinetic parameters. The primer was further extended by the addition of a ribonucleotide complementary to the nucleobase adjacent to C or A (the nucleobase opposite which 8-OH-GTP was inserted). Thus, the incorporation of 8-OH-GTP did not completely inhibit further RNA chain elongation. 8-OH-GTP was also incorporated opposite C and A by human RNA polymerase II. These results suggest that 8-OH-GTP in the nucleotide pool can cause the formation of oxidized RNA and disturb the transmittance of genetic information.

Numerous mutations are found in subacute sclerosing panencephalitis (SSPE) viruses, and the M gene is the gene most commonly affected. In some SSPE viruses, such as the MF, Osaka-1, Osaka-2, and Yamagata-1 strains, translation of the M protein is complicated by a transcriptional defect that leads to an almost exclusive synthesis of dicistronic P-M mRNA. To understand the molecular mechanisms of this defect, we sequenced the P gene at the P-M gene junction for several virus strains and probed the involvement of several mutations in the readthrough region via their expression in measles virus minigenomes containing different sequences of the P-M gene junction and flanking reporter genes. The deletion of a single U residue in the U tract of the Osaka-1 strain (3′-UAAUAUUUUU-5′) compared with the consensus sequence resulted in a marked reduction of the expression of the downstream reporter gene. In addition, the expression of the downstream gene was markedly decreased by (i) the substit...

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (Cre) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long Cre U3 snoRNA was immunoprecipitated by anti-γ-mpppN antisera, but not by anti-m 2,2,7 G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged Cre U3 snoRNA gene constructs were expressed in Cre cells. Results of chemical and enzymatic structure probing of Cre U3 snoRNA in solution and of DMS modification of Cre U3 snoRNA under in vivo conditions revealed that the twohairpin structure of the 5′-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5′-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented doublestranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

This study aims to determine the importance of conserved GDN motif in domain III and GXGXG motif in domain VI in Nipah virus (NiV) L protein. Four mutated L genes produced in an earlier study were inserted individually into plasmid pCITE. Optimised transfection protocol was successful in transfecting these plasmids, two helper plasmids (coding for N and P protein), NiV minigenome containing chloramphenicol acetyltransferase (CAT) reporter gene and T7 promoter. Successful in vitro transcription/translation in the NiV minireplicon system was monitored by CAT expression. In conclusion, GXGXG motif was important in the NiV minireplicon system but change of GDN motif does not affect L protein.

Background The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. Results We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has...

Most recently, an outbreak of severe pneumonia caused by the infection of 2019-nCoV, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. Since there is no crystallographic structure of RdRp is available, so, here, we present the 3-dimensional structure of the 2019-nCoV nsp12 polymerase using a computational approach. nsp12 of 2019-nCoV possesses an architecture common to all viral polymerases as well as a large N-terminal extension. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity, and acts as a template for the design of novel antiviral therapeutics. Besides, the experimental s...

Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements.

Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors σ F , σ E , σ G , and σ K coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be σ K dependent, while P2 and P3 were proposed to be σ E dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens . With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either ...

Pro-K is the inactive precursor of K , a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-K was present in the membrane fraction. The rest of the pro-K was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the K was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-E was analyzed. Pro-K was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-K did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-K processing in the mother cell. Furthermore, pro-K associated with the membrane when overproduced in vegetative cells. Overproduction of pro-K in sporulating cells resulted in more pro-K in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-K was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-K. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-K and K from other cell constituents. After salt removal, K , but not pro-K , reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-K to the membrane, where it may interact with the processing machinery.

Pro-ς K is the inactive precursor of ς K , a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis . Upon subcellular fractionation, the majority of the pro-ς K was present in the membrane fraction. The rest of the pro-ς K was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ς K was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-ς E was analyzed. Pro-ς K was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-ς K did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-ς K processing in the mother cell. Furthermore, pro-ς K associated with the membrane when overproduced in vegetative cells. Ov...

Trypanosoma cruzi is a kinetoplastid parasite that causes Chagas disease. Trypanosomes are unusual organisms in many aspects of its genetics and molecular and cellular biology and considered a paradigm of the exception of the rule in the eukaryotic kingdom. The complete genome sequence of T. cruzi was published in 2005, thus, providing a major tool to the understanding of several of his unusual aspects. However, with so many different mechanisms between the parasite and its mammalian host there is still a lack of availability of effective antiparasitic drugs or disease treatments, specially in the chronic phase. This review highlights the fundamentals of the fascinating genetics and genomics of T. cruzi with emphasis in the differential mechanisms that could provide interesting therapeutic targets.

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of Aplc5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p-, the INT promoter (pI), several sites in the b2 region, the mis promoter, the oop promoter (or po), and pro. Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the CIII-tL region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites.

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of fu...

Long polar fimbriae 1 (Lpf1) of Escherichia coli O157:H7 is a tightly regulated adhesin, with H-NS silencing the transcriptional expression of the lpf1 operon while Ler (locus of enterocyte effacement-encoded regulator) acts as an antisilencer. We mapped the minimal regulatory region of lpf1 required for H-NS- and Ler-mediated regulation and found that it is 79% AT rich. Three putative sites for H-NS binding were identified. Two of them, named silencer regulatory sequence 1 (SRS1) and SRS2, are located on a region that covers both of the lpf1 promoters (P1 and P2). The third putative H-NS binding site is located within the lpfA1 gene in a region extending from +258 bp to +545 bp downstream of ATG; however, this site does not seem to play a role in lpfA1 regulation under the conditions tested in this work. Ler was also found to interact with Ler binding sites (LBSs). Ler binding site 1 (LBS1) and LBS2 are located upstream of the two promoters. LBS1 overlaps SRS1, while LBS3 overlaps ...

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (,7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

The Sendai virus P and L proteins, the viral RNA polymerase, and the nucleocapsid protein, NP, synthesized in a transient mammalian expression system support the replication of Sendai virus defective interfering particle (DI) genome RNA in vitro. We have shown that the measles virus nucleocapsid protein, N, can substitute for the Sendai NP protein in genome synthesis. The chimeric product nucleocapsids, which contained Sendai RNA encapsidated with measles N protein, were atypical since they were sensitive to micrococcal nuclease digestion, unlike wild-type Sendai or measles nucleocapsids. The utilization of measles N protein required the endogenous Sendal virus RNA P01ymerase, since DI nucleocapsids free of poiymerase were not replicated. Although both Sendai virus NP and P proteins and measles N and P proteins formed complexes when they were coexpressed, sedimentation analysis showed that measles N protein self-assembled and did not form a complex when expressed with the Sendai P protein. Furthermore, when the Sendai P-L polymerase complex was provided separately, measles N protein alone synthesized DI genome RNA in the absence of Sendai P protein. These data suggest that the self-assembled form of measles N protein functions in Sendai DI genome synthesis.

Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975-6980, 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr 1 Ser 2 Pro 3 Thr 4 Ser 5 Pro 6 Ser 7 . In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.

A series of E. coli promoters made of the consensus -35 and -10 hexamers separated by 17 bp spacer with variously located bending dTn dAn, n = 5 or 6, sequences was constructed and cloned into the plasmid pDS3. Electrophoretic gel mobilities of restriction fragments containing these promoters correlated with the number of the T tracts encoded in the promoter sequences. The open complexes formed by E. coli RNA polymerase on promoters containing the T-(-34... -38) tract exhibited gel retardation indicative of their different gross geometry. The strength of these promoters measured in vivo in relation to an internal transcriptional standard was shown to be significantly lower than that of the group without the T5(-34...-38) tract. Within both these groups the promoters with two T6 tracts in the spacer, aligned in phase with the B-DNA helix repeat, had lower transcriptional activity, while the T6 tract encoded in the -7... -2 promoter region apparently had no influence on the strength of the respective promoters.