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TDP-43 is a major pathological protein in sporadic and familial amyotrophic lateral sclerosis (ALS) and mediates mRNA fate. TDP-43 dysfunction leads to causes progressive degeneration of motor neurons, the details of which remain elusive. Elucidation of the molecular mechanisms of RNA binding could enhance our understanding of this devastating disease. We observed the involvement of the glycine-rich (GR) region of TDP-43 in the initial recognition and binding of G-quadruplex (G4)-RNA in conjunction with its RNA recognition motifs (RRM). We performed a molecular dissection of these intramolecular RNA-binding modules in this study. We confirmed that the ALS-linked mutations in the GR region lead to alteration in the G4 structure. In contrast, amino acid substitutions in the GR region alter the protein structure but do not void the interaction with G4-RNA. Based on these observations, we concluded that the structural distortion of G4 caused by these mutations interferes with RRM recruitment and leads to TDP-43 dysfunction. This intramolecular organization between RRM and GR regions modulates the overall G4-binding properties. [ABSTRACT FROM AUTHOR]

Outside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization). The natural hosts of enterobacterium Escherichia coli are warm-blooded animals, but even outside hosts, E. coli can survive even under stressful environments. On earth, the most common organic materials to be used as nutrients by E. coli are plant-derived components, but up to the present time, the genetic system of E. coli for plant utilization is poorly understand. In the course of gSELEX screening of the regulatory targets for hitherto uncharacterized TFs, we identified in this study the involvement of the IclR-family YiaJ in the regulation of about 20 genes or operons, of which the majority are related to the catabolism of plant-derived materials such as ascorbate, galacturonate, sorbitol, fructose and fructoselysine. Therefore, we propose to rename YiaJ to PlaR (regulator of plant utilization). [ABSTRACT FROM AUTHOR]

The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as protein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly compacted than that in the exponential phase. In addition to factor for inversion stimulation (Fis), histone-like nucleoid structuring protein (H-NS), heat-unstable nucleoid protein (HU) and integration host factor (IHF) here we have identified, three new candidates of E. coli nucleoid, namely DNA-binding protein from starved cells (Dps), host factor for phage Qβ (Hfq) and suppressor of td- phenotype A (StpA). Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli. [ABSTRACT FROM AUTHOR]

Eukaryotic RNA polymerase II (Pol II) transcription apparatus is a multi-protein complex consisting of the RNA polymerase II core enzyme (12 subunits), general transcription factors, the mediator, and some other specific accessory factors with regulatory functions. After genome sequencing was completed, the fission yeast Schizosaccharomyces pombe was recognized as a good model organism to study the Pol II transcription apparatus, because most genetic methods developed with the budding yeast Saccharomyces cerevisiae are applicable but the genetic systems of Sch. pombe, including transcription, are closer to those in higher eukaryotes. Recent studies on components of the Sch. pombe basal transcription machinery not only revealed a number of properties common in other eukaryotes but also illuminated some features unique to Sch. pombe. Convergence of information from both yeasts will provide us with a more general understanding of eukaryotic transcription. [ABSTRACT FROM AUTHOR]

The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits. Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic α subunit, which plays a key role in RNA polymerase assembly. Together with the Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2–Rpb3–Rpb11) which corresponds to the the α2β assembly intermediate of prokaryotic RNA polymerase. For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S. pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3. The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S. pombe cell extracts. The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants. We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription. [ABSTRACT FROM AUTHOR]

Variation in the DNA sequence of the 10 kDa prolamin gene family within the wild rice species Oryza rufipogon was probed using the direct sequencing of PCR-amplified genes. A comparison of the nucleotide and deduced amino-acid sequences of eight Asian strains of O. rufipogon and one strain of the related African species O. longistaminata is presented. [ABSTRACT FROM AUTHOR]

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. Enzyme reconstitution experiments indicated that all three P proteins are required for RNA synthesis in vitro (Kobayashi, et al. Virus Res 22, 235-245, 1992). Nuclear extracts of HeLa cells infected with three kinds of the recombinant vaccinia virus, each carrying one of the three P protein cDNAs, exhibited the activity of complete replication, that is, vRNA-sense RNA-directed and cRNA-sense RNA-directed RNA synthesis in the absence of primers. The nuclear extract from cells singly infected with the virus carrying PB1 cDNA exhibited a significant level of model v-sense RNA-directed RNA synthesis activity. Thus we conclude that PB1 is the catalytic subunit of influenza virus RNA polymerase and that under certain conditions, PB1 alone is able to catalyze RNA synthesis in vitro. [ABSTRACT FROM AUTHOR]

DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV 1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV 1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV 1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted. [ABSTRACT FROM AUTHOR]

Influenza virus M protein has been shown to inhibit viral RNA transcription, and in this study the epitopes on M critical for this function were localized. When a battery of 15 monoclonal anti-M antibodies were reacted with chemically cleaved fragments of M on a western blot, five distinct banding patterns were observed. A representative antibody was selected from each banding group, and its ability to reverse M-effected transcription inhibition was measured. From these data, the sites on M critical for transcription inhibition were deduced. It appears now that the regions on M in the vicinity of amino acid residues ♯70 and ♯140 are critical for inhibition. Furthermore, by taking into account the hydropathicity and secondary structure, it is hypothesized that amino acids ♯70 and ♯140 are physically close together in the final three-dimensional conformation of M protein and that the residues in between form a loop and are thus removed from the functional site. [ABSTRACT FROM AUTHOR]

cDNAs for genome RNAs of influenza virus A/PR/8/34 were cloned, and portions containing the ATG for initiation codon of translation were inserted into the 5′ leader sequence of the chloramphenicol acetyltransferase (CAT) gene in a pSV2cat vector. When transfected cells were super-infected with influenza virus, the CAT activity was found to vary in a time-dependent fashion: A construct containing a cDNA segment for the nonstructural (NS) protein directed the highest activity during the early stage of infection, while a construct containing a cDNA segment for the neuraminidase (NA) directed the highest activity during the late stage of infection. This time-dependent variation in the CAT activity is in good agreement with that of the synthesis rate of respective viral proteins in infected cells. We propose that the translational efficiency of viral mRNA is subjected to temporal control following viral infection, although viral protein synthesis itself is regulated primarily at the level of mRNA synthesis. [ABSTRACT FROM AUTHOR]

The regulation of gene expression was studied for the Escherichia coli rpoBC operon, which includes the genes, rpoB and rpoC, for the β and β′ subunits of RNA polymerase, and rplJ and rplL, for the two proteins, L10 and L7/12, of the 50S ribosome. The gene organization agrees well with the accumulated observations indicating the coordinate synthesis of RNA polymerase and ribosomes under various growth conditions for wild-type E. coli cells. On the other hand, the differential regulation of the two essential components observed under restrictive growth conditions, after addition of various drugs or with certain mutants, in particular those carrying mutations in the RNA polymerase genes, was found to take place through two novel regulation systems: The transcriptional termination at an internal attenuation site and the two autogenous and post-transcriptional controls, being specific for the two ribosomal protein genes and the two RNA polymerase subunit genes, respectively. The majority of the transcription initiated from the promoter rpoP terminates at an attenuator site between the promoter-proximal rplJL and the promoter-distal rpoBC genes. The frequency of the attenuation seems to control the relative level of RNA polymerase synthesis to that of ribosomes. The expression of rpoBC genes is subject to an autogenous regulation, in which both RNA polymerase holoenzyme and αβ complex function as regulatory molecules with repressor activity. The autogenous regulation was found to operate at post-transcriptional step(s), probably at the level of translation. During the study on the regulation of RNA polymerase synthesis, we noticed that the rpoBC operon contained another autogenous regulation circuit, in which the synthesis of L10 and L7/12 was specifically repressed by the L10-L7/12 complex. Molecular mechanisms and physiological meanings of the novel regulations are discussed. [ABSTRACT FROM AUTHOR]