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At least 379 records · Page 21Linked to original sources
PubMed1988

Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the L protein.

The sequence of the gene encoding the L protein of the human parainfluenza 3 virus was determined by direct dideoxy sequence analysis of the genomic 50 S RNA and confirmed by molecular cloning and sequence analysis of recombinant clones. A series of three overlapping clones was generated by primer extension using genomic 50 S RNA as the template. These clones originate within the 5' end of the hemagglutinin-neuraminidase gene, span the entire L gene, and extend into the extracistronic 5' end of the viral RNA. The L gene extends 6755 nucleotides (inclusive of the putative transcription initiation and polyadenylation signal sequences) and encodes a protein consisting of 2233 amino acids (MW 255,812). There are 44 nucleotides downstream of the putative polyadenylation signal sequence which may represent a negative-strand leader. The complementary sequence of the extracistronic region is nearly identical to the 3' end of the viral RNA. Thirty-three of the first thirty-nine nucleotides of the 3' ends of the plus and minus strands are conserved. Comparison of amino acid sequence homology with other paramyxoviral L proteins indicates a high degree of sequence conservation with Sendai virus (62%) and Newcastle disease virus (28%). In addition, four smaller regions were identified which shared extensive homology with the L protein of vesicular stomatitis virus, a member of the Rhabdoviridae family.

Amino Acid Sequence↗
PubMed2003

The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mRNA synthesis.

Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a "body" part of variable size, which are derived from the 5'- and 3'-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus subgenomic mRNA synthesis.

Antisense Elements (Genetics)↗
PubMed1993

Self-splicing of the Tetrahymena pre-rRNA is decreased by misfolding during transcription.

RNA processing depends in part on the ability of nascent transcripts to fold into the desired conformation. Self-splicing of the group I intron from Tetrahymena was used to assess the folded state of preribosomal RNA transcripts when synthesized in vitro. A simple method for isolating nondenatured RNA from a T7 RNA polymerase reaction was tested. The intron alone is fully active when transcribed at 30 degrees C, suggesting that the active structure is both kinetically and thermodynamically favored. Longer precursor RNAs, however, were less than completely active in self-splicing. Full activity, as judged by both the initial rate and the extent of product formation, was restored by brief incubation at 95 degrees C and rapid cooling in the presence of magnesium ion. This result did not depend on the length of the precursor RNA in any simple way, but correlated loosely with the presence of intact exon domains. When transcribed in the absence of cellular proteins, a significant portion of the pre-RNA appears to be trapped in a conformation that does not readily undergo the first step of splicing.

Animals↗
PubMed1994

Determinants of the p28 cleavage site recognized by the first papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus polymerase gene is 22 kb in length with the potential to encode a polyprotein of approximately 750 kDa. The polyprotein has been proposed to encode three proteinase domains which are responsible for the processing of the polyprotein into mature proteins. The proteolytic activity of the first proteinase domain has been characterized and resembles the papain family of cysteine proteinases. This proteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein designated p28. To identify the cleavage site of this papain-like cysteine proteinase, we isolated the peptide adjacent to p28 and determined the amino terminus sequence by Edman degradation reaction. We report that proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleavage site, we introduced a total of 42 site-specific mutations at the residues spanning the P5 to P3' positions and assessed the effects of the mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position or Arg-246 at the P2 position resulted in a dramatic decrease of proteolytic activity, and the mutations of Arg-243 at P5 position also led to considerable reduction in p28 cleavage. In contrast, the substitutions of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 (P2'), and Pro-250 (P3') had little or no effect on the amount of p28 that was released. This work had identified Gly-247-Val-248 as the cleavage site for the release of p28, the amino-terminal protein of the murine coronavirus polymerase polyprotein. Additionally, we conclude that the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murine coronavirus.

Amino Acid Sequence↗
PubMed2003

Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.

We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.

Base Sequence↗
PubMed1999

Reevaluation of nucleotide sequences of wild-type and attenuated polioviruses of type 3.

Published sequences of wild-type and attenuated Sabin strains of type 3 poliovirus (Leon/37 and Leon 12a(1)b) were derived from cDNA clones. Recent direct sequencing of Sabin 3 RNA showed that it differed from the published sequence in at least two sites. Here results of direct sequencing of genomes of three independently re-derived sub-strains of attenuated Sabin 3 poliovirus used for oral poliovirus vaccine (OPV) production in addition to the most widely used Pfizer sub-strain are reported. The results showed that all four sub-strains of attenuated type 3 poliovirus contain unique patterns of mutations. Two stocks of the wild-type progenitor Leon/37 strain were also sequenced. Analysis of the two samples of Leon/37 virus showed that one of them is much closer to the Sabin 3 strain, and is an intermediate product of the attenuation process. In addition, we created genetically engineered constructs which contained some of the mutations suspected for their possible role in neurovirulence, and tested them in monkeys and in transgenic mice sensitive to poliovirus. The results suggested that none of them increased neurovirulence of the virus, but some may improve virus replication. Therefore the only mutation occurring in Sabin 3 under vaccine production conditions that appears to affect neurovirulence of the virus is the well known U-->C reversion at nucleotide 472.

Animals↗
PubMed2000

Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.

Amino Acid Sequence↗
PubMed2003

The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes.

Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.

5' Untranslated Regions↗
PubMed2002

The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases.

In vitro, bacteriophage N4 virion RNA polymerase (vRNAP) recognizes in vivo sites of transcription initiation on single-stranded templates. N4 vRNAP promoters are comprised of a hairpin structure and conserved sequences. Here, we show that vRNAP consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini-vRNAP). Biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specificity and lack of extensive sequence similarity to other DNA-dependent RNA polymerases, mini-vRNAP is related to the family of T7-like RNA polymerases.

Amino Acid Motifs↗
PubMed2001

Unnatural base pairs for specific transcription.

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

Base Pairing↗
PubMedDate not supplied

[RNA synthesis by T7 RNA polymerase supported primer extension].

Transcription of RNA molecules from synthetic DNA templates with T7 RNA polymerase is a common procedure for the preparation of long RNA molecules. However, enzymatic synthesis does not allow for site-specific incorporation of modified nucleotides. RNA synthesis by chemical methods on the other side can satisfy this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed to combine both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end we have chemically synthesised a fluorescein labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We show that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesised by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.

Base Sequence↗
PubMed1986

Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations.

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C1 in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T1 RNase oligonucleotide fingerprinting. Values ranged from less than 0.04 X 10(-2) to 4.5 X 10(-2) substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10(-2) s/nt/yr have been measured for the RNA region that encodes VP1-VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C1 isolates, and they are compared with the two previously known sequences of FMDV C1 (C-S8 and C1-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C1 outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs ('evolving quasispecies') as the mode of evolution of this agent.

Amino Acid Sequence↗
PubMed1992

Binding of TFIIIA to derivatives of 5S RNA containing sequence substitutions or deletions defines a minimal TFIIIA binding site.

The repetitive zinc finger domain of transcription factor IIIA binds 5S DNA and 5S RNA with similar affinity. Site directed mutagenesis of the Xenopus borealis somatic 5S RNA gene has been used to produce a series of derivatives of 5S RNA containing local sequence substitutions or sequence deletions. Gel mobility shift analyses of the binding of TFIIIA to these altered 5S RNAs revealed that all three of the helical stems of the 5S RNA secondary structure are required for binding. TFIIIA was observed to bind with normal affinity to RNAs lacking 12 nucleotides at either the loop c or loop e/helix V regions of 5S RNA, as well as to a double mutant containing both deletions. The secondary structure of the resulting 96-nucleotide RNA, studied using structure-specific ribonucleases, was found to resemble the central portion of 5S RNA.

Animals↗
PubMed1994

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of beta', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of beta'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of beta'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different rho-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of the genes encoding proline tRNA(UGG) and threonine tRNA(GGU) and consensus promoter model of Thermococcus celer.

The nucleotide sequences of the genes encoding tRNA(Pro)(UGG) and tRNA(Thr)(GGU) from the extremely thermophilic archaeon (archaebacterium) Thermococcus celer have been determined. A consensus promoter model was deduced from the comparison of the upstream regions of several stable RNA genes with S1-mapped promoter regions of genes coding for ribosomal proteins and DNA-dependent RNA polymerase components.

Archaea↗
PubMed1992

Structure of transcription elongation complexes in vivo.

The opening of duplex DNA in the elongation phase of transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in transcription occurs. Single-stranded DNA in the transcription bubble was identified by its reactivity with potassium permanganate (KMnO4). The elongation structure in vivo was similar to that of transcription complexes made in vitro with some differences. The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.

Base Sequence↗
PubMed1988

Identification of a repeated sequence in the genome of the sea urchin which is transcribed by RNA polymerase III and contains the features of a retroposon.

A repeated sequence element which is located about 200 nucleotides upstream from the protein-coding portion of the muscle actin gene (probably within a large 5' intron) in the genome of the sea urchin, Strongylocentrotus purpuratus has been characterized, and shown to contain the sequence features which indicate that it has been transposed by means of an RNA intermediate. This retroposon-like sequence, SURF1-1, is a member of a family which is dispersed and repeated about 800 times in the genome, referred to as SURF1 (sea urchin retroposon family 1). In vitro transcription of this sequence by RNA polymerase III defines a 300 nucleotide transcription unit which is bounded by a short direct repeated sequence. The 3' end of this unit contains a simple 21 nucleotide A+T-rich sequence characteristic of retroponons, and a consensus B box portion of an internal RNA polymerase III promotor is located 60 to 80 nucleotides downstream from the two sites of transcription initiation. This sequence also contains a 40 nucleotide region that is related to several tRNA sequences (containing the B box), and a 79 nucleotide sequence which is homologous to a repeated sequence previously shown to be present within the 3' untranslated portions of the Spec1 and Spec2 mRNAs of this species (1). Analysis of transcripts of this sequence family in RNA from several embryonic stages indicates that its expression is highest at 11 hours postfertilization (about 128 cells) and drops as development proceeds. Furthermore, most or all, transcription of this sequence family in nuclei isolated from 11 hour embryos is by RNA polymerase III, and is from the same strand which is transcribed in vitro.

Actins↗