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At least 289 records · Page 16Linked to original sources
PubMed1995

The positively acting amdA gene of Aspergillus nidulans encodes a protein with two C2H2 zinc-finger motifs.

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdl66 and amdl666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles. The wild-type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880-amino-acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains. The amdA7 semi-dominant gain-of-function mutation results in a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5' region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc-finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.

Acetates↗
PubMed1997

Identification of the active site histidine in the corrinoid protein MtrA of the energy-conserving methyltransferase complex from Methanobacterium thermoautotrophicum.

The energy-conserving corrinoid-containing MtrA-H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group. EPR spectroscopic evidence has recently been presented for a histidine residue as a cobalt ligand of the cobamide [Harms, U. & Thauer, R. K. (1996a) Eur. J. Biochem. 241, 149-154]. This active site histidine was now identified by site-directed mutagenesis to be His84 in the MtrA sequence that contains three histidines. This result was substantiated by sequence comparison of MtrA from M. thermoautotrophicum, Methanococcus jannaschii, and Methanopyrus kandleri and of MtxA from Methanosarcina harkeri showing that only His84 is conserved. For comparison, the DNA sequences of the mtrEDCBAGH operon in M. kandleri and of the mtxXAH operon in M. barkeri were determined.

Amino Acid Sequence↗
PubMed1991

Interaction of C-terminal sequences of human immunodeficiency virus reverse transcriptase with template primer.

We have raised a rabbit monospecific antibody (designated C2003) against a synthetic peptide (CTP66) derived from a conserved sequence in the C-terminal portion of the p66 component of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (DeVico, A.L., Copeland, T.D., Veronese, F.D., Oroszlan, S., Gallo, R. C., and Sarngadharan, M. G. (1989) AIDS Res. Hum. Retroviruses 5, 51-60). This antibody directly inhibits the polymerase activity of HIV-1 RT and of RTs from a variety of retroviruses. HIV-1 RT is protected from this inhibition by preincubation of the enzyme with template primer prior to treatment with the antibody. Such protection is abrogated when the pretreatment is conducted under conditions of high ionic strength. Kinetic studies showed that the antibody-mediated inhibition is competitive with respect to template primer concentration. These results indicate that C2003 antibody acts to interfere with the template binding function of the enzyme and further indicates that conserved residues recognized by the antibody may be directly involved in this function.

Amino Acid Sequence↗
PubMed1993

Direct cloning and expression of PCR amplified DNA and RNA sequences: application to the hepadnaviruses nucleocapsid proteins.

Gene amplification may benefit from the construction of primers that augments the speed at which cloning and protein expression proceeds. Such primers include EcoRI or HindIII linkers as well as an in phase initiation or termination codon. PCR was carried out directly from viral particles of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) without DNA purification and from RNA extracted from WHV infected liver. Amplified products were directly cloned in the pKK223-3 expression vector under the control of the tac promoter. The characterization of the recombinant clones expressing the nucleocapsid protein (C protein) was done by direct incubation of the filter with 125I-labelled anti-HBc and confirmed by radioimmunoassay and Western-blot analysis. This procedure allows easy selection of recombinant clones expressing a given protein and could be applied to many other genes.

Animals↗
PubMed2000

Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3' cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3' single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3'-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

Hepacivirus↗
PubMed1997

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Amino Acid Sequence↗
PubMed1989

Mutagenesis analysis of a self-cleaving RNA.

The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage.

Base Sequence↗
PubMed1993

RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.

The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.

Base Sequence↗
PubMed1998

Terminator-specific recycling of a B1-Alu transcription complex by RNA polymerase III is mediated by the RNA terminus-binding protein La.

Efficient synthesis of many small abundant RNAs is achieved by the proficient recycling of RNA polymerase (pol) III and stable transcription complexes. Cellular Alu and related retroposons represent unusual pol III genes that are normally repressed but are activated by viral infection and other conditions. The core sequences of these elements contain pol III promoters but must rely on fortuitous downstream oligo(dT) tracts for terminator function. We show that a B1-Alu gene differs markedly from a classical pol III gene (tRNAiMet) in terminator sequence requirements. B1-Alu genes that differ only in terminator sequence context direct differential RNA 3' end formation. These genes are assembled into stable transcription complexes but differ in their ability to be recycled in the presence of the La transcription termination factor. La binds to the nascent RNA 3' UUUOH end motif that is generated by transcriptional termination within the pol III termination signal, oligo(dT). We found that the recycling efficiency of the B1-Alu genes is correlated with the ability of La to access the 3' end of the nascent transcript and protect it from 3'-5' exonucleolytic processing. These results illuminate a relationship between RNA 3' end formation and transcription termination, and La-mediated reinitiation by pol III.

Alu Elements↗
PubMed1993

Relief of triple-helix-mediated promoter inhibition by elongating RNA polymerases.

We have characterized triple-helix-mediated inhibition of an artificial bacteriophage promoter with respect to relief of inhibition by incoming RNA polymerases that initiate upstream or downstream from the operator sequence. Whereas oligonucleotide-directed triple-helix formation inhibits the test promoter, promoter activity is restored when the triple-helical complexes are disrupted by transcription of either strand of the homopurine operator sequence. The degree of relief from inhibition is related to the frequency of operator transcription. These observations demonstrate that this artificial repressor-operator complex is subject to antagonism by cis elements (other promoters) acting at a distance. Such antagonism might also arise between certain natural transcriptional control regions. Our results suggest that the efficiency of artificial repressors based on triple-helix formation may be limited by transcriptional activity in the gene control region.

Bacteriophage T7↗
PubMed1999

Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation.

Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobacco plants has suggested that an RNA-DNA interaction could trigger de novo methylation of PSTVd transgene sequences. Using the genomic sequencing technique, the methylation pattern associated with the RNA-directed DNA methylation process has been characterized. Three different PSTVd transgene constructs all showed a similar pattern of methylation. Most of the cytosines at symmetrical as well as non-symmetrical positions appeared to be methylated in both DNA strands of the viroid sequences. Heavy methylation was mostly restricted to the viroid cDNA sequences. Flanking DNA regions immediately adjacent to the viroid cDNA displayed a lower but significant level of cytosine methylation. The observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act as a strong and highly specific signal for de novo DNA methylation. These data also confirmed that de novo methylation was not limited to canonical CpG and CpNpG sites, but can also involve all the cytosine residues located in the genomic region where the RNA-DNA interaction takes place.

Base Sequence↗
PubMed1993

Effects of multiple mutations at the conserved TATA sequence of bacteriophage SP6 promoter on transcription efficiency.

Mutations of A<==>T were introduced individually and multiple to TATA from -4 to -1 of the phage SP6 promoter and their effects on transcription initiation efficiency measured in vitro. All 15 mutants tested were less active than the wild type. Mutation at -4T nearly abolishes promoter activity independent of other changes, and alteration at -3A reduces promoter activity substantially. On the other hand, effects of mutations at -2T and -1A depend on other changes, suggesting their role should be associated with neighboring base pairs. These results suggest that -4T and -3A are involved in SP6 RNA polymerase binding and -2T and -1A are involved in DNA unwinding. This bipartite role of the SP6 promoter TATA contrasts with the single role of T7 promoter TATA on DNA unwinding. The polymerase binding region extends further downstream in the SP6 promoter than in the T7 promoter.

Bacteriophages↗
PubMed2005

The complete genome sequence, organization and affinities of carrot red leaf virus.

A sequence of 5723 nucleotides (GenBank accession number: AY695933) is reported for the RNA genome of an isolate of Carrot red leaf virus (CtRLV). The sequence is predicted to contain six large open reading frames and non coding sequences of 28 nucleotides at the 5' end, 110 nucleotides at the 3' end, and 215 nucleotides between the two main blocks of coding sequences. The 5' coding region encodes two polypeptides with calculated molecular masses (Mr) of 28.6 kDa (P0) and 68.2 kDa (P1) that overlap in different reading frames. Circumstantially, the third ORF in the 5' block is putatively translated by frameshift read-through to yield a polypeptide (P1 + P2) with a calculated Mr of 116.9 kDa. Frameshifting is predicted at a "shifty" sequence (GGGAAAC; nt 1523-1529) also found in most members of the genus Polerovirus. The C-terminal region of the 116.9 kDa polypeptide includes the consensus sequence for the viral RNA-directed RNA polymerase. The 3' block of coding sequence defines three putative polypeptides of: 23.0 kDa (P3), 21.3 kDa (P4, in a different reading frame) and 77.2 kDa (P3 + P5, by read-through of P3) respectively. From the genome structure of CtRLV, it is suggested that this virus belongs to the genus Polerovirus, rather than either the genus Luteovirus or the genus Enamovirus.

5' Flanking Region↗
PubMed1996

Development of a dual target-PCR for detection and characterization of measles virus in clinical specimens.

A polymerase chain reaction (PCR) method was developed for detecting measles virus (MV) RNA in a variety of clinical samples using primer pairs in the nucleocapsid (N) and matrix (M) genes in one reaction (dual target-PCR). The dual target-PCR detected MV RNA in tissue culture fluid containing 1 TCID50 of the MV Loss strain, and was as sensitive as a single target-PCR. Specificity was confirmed by the failure of the dual target-PCR to amplify products from the infected tissue culture containing related paramyxoviruses. Thirty-two of 35 (91.4%) samples collected from 23 patients with confirmed measles infection by the detection of measles specific IgM were found to be positive for MV RNA by PCR. Direct sequencing of the PCR amplicons revealed three different genotypes among the MV strains that were detected in 12 patients. The dual target-PCR method is suitable for the diagnosis of measles infection and, based on the sequencing of the PCR product DNA, for investigating the molecular epidemiology of MV strains.

Adult↗
PubMed1992

Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition.

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."

Affinity Labels↗
PubMed1996

Large RNA segment of Dugbe nairovirus encodes the putative RNA polymerase.

The nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.

Amino Acid Sequence↗
PubMed1995

sigma E changed to sigma B specificity by amino acid substitutions in its -10 binding region.

The association of a sigma factor (sigma) with RNA polymerase in bacteria determines its specificity of promoter utilization. To identify amino acid residues in sigma E from Bacillus subtilis that determine the specificity of its interaction with the nucleotides at the -10 region of its cognate promoters, we tested whether base pair substitutions in the -10 region of a sigma B-dependent promoter could signal its utilization by sigma E-RNA polymerase. We found that a combination of base pair substitutions at positions -15 and -14 of the sigma B-dependent ctc promoter resulted in its utilization by sigma E-RNA polymerase in vivo. We also found that the combination of two amino acid substitutions at positions 119 and 120 in sigma E changed its specificity for promoter utilization, resulting in a sigma factor that directed transcription from the sigma B-dependent ctc promoter, but not from sigma E-dependent promoters. These results suggest that amino acid residues at positions 119 and 120 determine, at least in part, the specificity of interactions between sigma E and the nucleotides in the -10 region of its cognate promoters.

Amino Acid Sequence↗
PubMed1990

An RNA polymerase II promoter containing sequences upstream and downstream from the RNA startpoint that direct initiation of transcription from the same site.

The gfa gene encodes glial fibrillary acidic protein (GFAP), an intermediate-filament protein expressed primarily in glial cells. We have used in vitro transcription studies to show that the basal level of transcription of the human gene encoding GFAP is controlled by two distinct initiators--i.e., promoter elements that direct transcription from a specific start site. One initiator is located about 25 base pairs upstream from the transcription start site, contains a TATA box, and apparently acts together with a sequence found around the transcription start site. The other initiator is located between +11 and +50 bp downstream from the transcription start site. Most of this second region overlaps with the protein-encoding sequence, which starts at bp +17. The sensitivity of transcription to alpha-amanitin indicates that both initiators are used by RNA polymerase II.

Amino Acid Sequence↗