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At least 703 records · Page 39Linked to original sources
PubMed1999

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.

Animals↗
PubMed1999

Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viral RNA template during RNA replication.

The viral polymerase of influenza virus, a negative-strand RNA virus, is believed to polyadenylate the mRNAs by stuttering at a stretch of five to seven uridine residues which are located close to the 5' ends of the viral RNA templates. However, a mechanism of polyadenylation based on a template-independent synthesis of the poly(A) tail has not been excluded. In this report, we present new evidence showing the inherent ability of the viral polymerase to stutter at the poly(U) stretch of a viral RNA template during RNA replication. Variants which possess 1- to 13-nucleotide-long insertions at the poly(U) stretch have been identified. These results support a stuttering mechanism for the polyadenylation of influenza virus mRNAs.

Base Sequence↗
PubMed1985

Novel structure of a human U6 snRNA pseudogene.

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.

Bacteriophage lambda↗
PubMed1994

Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit.

It is now generally accepted that 16S and 23S ribosomal RNA play important roles in the decoding and peptidyl transferase activities of ribosomes. Despite their complex structures and numerous associated proteins it is possible that small domains of these rRNAs can fold and function autonomously, particularly those that appear devoid of protein interactions. One candidate for such a domain is the decoding region, located near the 3' end of 16S rRNA (Fig. 1a, b). Consistent with this hypothesis, aminoglycoside antibiotics that interact with the decoding region in 30S subunits interact with other RNAs in the absence of proteins. In addition, certain activities of self-splicing introns, at least superficially, resemble translational decoding. We report here that an oligoribonucleotide analogue of the decoding region interacts with both antibiotic and RNA ligands of the 30S subunit in a manner that correlates with normal subunit function. The activities of the decoding region analogue suggest that the intimidating structural complexity of the ribosome can be, to some degree, circumvented.

Anti-Bacterial Agents↗
PubMed1993

3'-terminal nucleotide sequences important for the accumulation of cowpea mosaic virus M-RNA.

The location of nucleotide sequences important in determining the extent of cowpea mosaic virus M-RNA accumulation in cowpea protoplasts has been analyzed by deletion mutagenesis of full-length cDNA clones from which infectious transcripts can be produced in vitro. The results suggest that cis-acting sequences which direct replication of M-RNA by B-RNA-encoded products are located within the 5'-terminal 524 nucleotides and the 3'-terminal 151 nucleotides. RNA secondary structure predictions for the 3'-terminal 151 nucleotides of both genomic RNAs (Eggen et al. (1989) Virology 173, 456-464) indicate that the terminal nucleotides form a stable secondary structure composed of a Y-shaped stem-loop and a simple A-U-rich stem-loop. The latter structure has been implicated in B-RNA replication. We have examined the role of the Y-shaped structure in M-RNA accumulation by site-directed mutagenesis of putative base-pairing combinations in the two minor stems. The results suggest that efficient replication is dependent on the formation of both of these minor stem structures.

Base Sequence↗
PubMed1994

Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis.

Sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral RNA replication and transcription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindbis virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein comprising two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of random linker insertion mutations in the nsP3-coding region and characterized selected viable mutants. Initially, 126 mutants containing insertions in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved-region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque phenotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3.39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutations localized to different regions near nsP3 residues 58 and 226, respectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from the library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand and subgenomic RNAs.

Amino Acid Sequence↗
PubMed2002

Role of the RNA polymerase sigma subunit in transcription initiation.

In bacteria, sigma subunits direct the catalytically competent RNA polymerase core enzyme to promoters. Recent advances in our understanding of bacterial RNA polymerase reveal that sigma subunits are intimately involved in all aspects of transcription initiation including promoter location, promoter melting, initiation of RNA synthesis, abortive initiation and promoter escape.

Amino Acid Sequence↗
PubMed1994

Polymerase chain reaction (PCR) as a diagnostic tool in HIV infection.

We set up a PCR laboratory for the diagnosis of HIV-1. Probably due to the variability of the HIV-genome, classical primers that performed well in some laboratories in the past, did not suffice for detection of HIV-1 strains in Belgian hospitals. Two new primer sets amplifying a fragment in the LTR-gag gene and in the env gene, which perform better on strains seen in Belgium, have been developed and evaluated. One primer set, conceived and evaluated on Belgian strains by the "Instituut voor Tropische Geneeskunde" in Antwerp, was also included. These three primer sets performed superior (92% sensitivity and 100% specificity on 24 samples) than the classical primers (83.5% sensitivity and 56% specificity on 21 samples). Together with a well-studied testing algorithm, they allow the reliable identification of the presence of the HIV-1 genome. To detect resistance of HIV-1 to reverse transcriptase (RT) inhibitors, we developed a set of two overlapping nested PCR primer sets and additional sequencing primers to amplify and sequence the total RNA or DNA RT gene using a direct cycle sequencing approach of the amplified fragment. Some clinical isolates were amplified and sequenced. In HIV-1 isolates from TIBO R82913-treated patients we identified two amino acid mutations (V108I and Y188L) involved in resistance (more than 100-fold reduced sensitivity). In an untreated patient we identified an amino acid variant (I/V 179D) involved in a 7-fold reduced sensitivity to TIBO. Several other amino acid variants, not involved in resistance, were detected in treated and untreated patients. Using this sequencing technique on cultured virus isolates we also observed in one TIBO-treated patient a differential selection among the strains of the original HIV-1 pool. From this patient we isolated and sequenced a completely TIBO sensitive HIV-1 strain after extensive cultivation in cord blood lymphocytes of the original TIBO resistant HIV-1 virus pool. We could however identify the resistant genotype after cultivation of this resistant HIV-1 virus pool on CEM cells. Our study revealed that sequencing investigations on emerging resistance should preferentially be done with uncultured patient samples since viral sequences and virus-drug sensitivities obtained from isolates cultured in vitro may not necessarily correspond to the sequences and sensitivities of the dominant strain in vivo.

Amino Acid Sequence↗
PubMed2005

New ligase-derived RNA polymerase ribozymes.

The search is underway for a catalytic RNA molecule capable of self-replication. Finding such a ribozyme would lend crucial support to the RNA World hypothesis, which holds that very early life-forms relied on RNA for both replicating and storing genetic information. We previously reported an RNA polymerase isolated from a pool of variants of an existing RNA ligase ribozyme. Here we report eight additional ligase-derived polymerase ribozymes isolated from this pool. Because each of them is a new potential starting point for further in vitro evolution and engineering, together they substantially enrich the set of candidates from which an RNA replicase ribozyme might eventually emerge.

Base Sequence↗
PubMedDate not supplied

Progress towards a higher taxonomy of viruses.

The current consensus view is that a higher hierarchical taxonomy of viruses cannot be established for two reasons. Firstly, viruses appear to be polyphyletic in origin, with several sets of viruses arising by different, independent routes at different times. Secondly, subsequent virus adaptation for survival in different host/vector combinations has involved the selective acquisition of additional genes by a process of cassette or modular evolution, with these additional gene modules coming from other viruses or host genetic material. Thus, depending on the gene product used for comparison, different phylogenetic relationships can be deduced. Further virus adaptation can arise by reassortment of segmented genomes, gene duplication, deletions, frameshift mutations, point mutations or de novo development of new gene products from existing, unused reading frames. The solution to the first objection is to place all viruses in a separate kingdom and assign the current viruses to several phyla that reflect these diverse origins. The solution to the second objection is to consider the core module of replication machinery as the major criterion on which to make the initial assignments to classes and orders. For RNA viruses, the major criterion is the sequence identity of the RNA-dependent RNA polymerase. Using this criterion, the positive strand RNA viruses can be assigned to five classes that correspond to the recently recognized supergroups of RNA viruses. These five classes contain four, three, three, three and one order(s) respectively. These fourteen orders contain 31 virus families (including 17 families of plant viruses) and 48 genera (including 30 genera of plant viruses). This approach confirms the separation of the alphaviruses and flaviviruses into two families, the Togaviridae and Flaviridae, but suggests that several other current taxonomic assignments, such as the pestiviruses, hepatitis C virus, rubiviruses, hepatitis E virus and arteriviruses, may be wrong. The coronaviruses and toroviruses appear to be distinct families in distinct orders, not distinct genera of the same family as currently classified. In addition, the luteoviruses are split into two families and apple chlorotic leaf spot virus appears not to be a closterovirus but a new genus of the Potexviridae. From an analysis of the polymerase dendrograms of the dsRNA viruses, it appears that they are not closely related to each other, but belong to four additional classes (Partitiviridae, Reoviridae, Birnaviridae and Cystoviridae) and one additional order (Totiviridae) of one of the classes of positive ssRNA viruses in the same subphylum as the positive strand RNA viruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2000

Mutually exclusive utilization of P(R) and P(RM) promoters in bacteriophage 434 O(R).

Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the P(RM) promoter and repress transcription from the divergent P(R) promoter. Several lines of evidence indicate that the 434 repressor activates initiation of P(RM) transcription by occupying a binding site adjacent to the P(RM) promoter and directly contacting RNA polymerase. The overlapping architecture of the P(RM) and P(R) promoters suggests that an RNA polymerase bound at P(R) may repress P(RM) transcription initiation. Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the P(RM) promoter and to the P(R) promoter. Consistent with this proposal, we show that the repressor cannot activate P(RM) transcription if RNA polymerase binds at P(R) prior to addition of the 434 repressor. However, unlike the findings with the related lambda phage, formation of RNA polymerase promoter complexes at P(RM) and at P(R) apparently are mutually exclusive. We find that the RNA polymerase-mediated inhibition of repressor-stimulated P(RM) transcription requires the presence of an open complex at P(R). Taken together, these results indicate that establishment of an open complex at P(R) directly prevents formation of an RNA polymerase-P(RM) complex.

Bacteriophage lambda↗
PubMed1998

Characterization of a gene encoding a single-subunit bacteriophage-type RNA polymerase from maize which is alternatively spliced.

Single-subunit RNA polymerases belonging to the T3/T7 bacteriophage family are thought to be common throughout eukaryotes. We report the isolation and characterization of a nucleus-encoded single-subunit RNA polymerase gene from maize. This gene is highly homologous to other single-subunit RNA polymerase genes from Arabidopsis, Chenopodium. yeast and Neurospora crassa involved in organellar transcription. Genomic Southern analysis reveals 10 to 15 hybridising fragments, suggesting that maize contains a small gene family. The isolated gene contains 19 exons and its genomic structure is highly conserved when compared to the three Arabidopsis homologues. Unlike the case in Arabidopsis, intron-12 of the maize bacteriophage-type RNA polymerase gene is alternatively spliced. Quantitative RT-PCR revealed that the resultant alternatively spliced transcript represents approximately 21 to 26% of the total polymerase mRNA in maize coleoptiles. The orthologous wheat bacteriophage-type RNA polymerase is also alternatively spliced and the intron exhibits 78% identity to maize intron-12. The conservation in alternative splicing between wheat and maize and its absence from Arabidopsis suggest a functional requirement for the alternatively spliced product.

Alternative Splicing↗
PubMed1995

A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.

Amino Acid Sequence↗
PubMed1996

Characterization of the core promoter of the Na+/K(+)-ATPase alpha 1 subunit gene. Elements required for transcription by RNA polymerase II and RNA polymerase III in vitro.

We have analyzed the core promoter element of the Na+/K(+)-ATPase alpha 1 subunit gene by means of an in vitro transcription system composed of a HeLa nuclear extract. 5'-deletion and 3'-deletion analyses revealed that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gene (-102 to -61), and that the 3' boundary of the minimal promoter element does not extend beyond +5. Analysis of linker-substitution mutations and point mutations revealed that the TATA-like sequence (-33 to -26) is required for upstream-sequence-dependent transcription whereas linker-substitution mutations and point mutations near +1 did not abolish transcription. The gene was found to be transcribed by RNA polymerase III when phosphocellulose column fractions were assayed. Deletion analysis mapped the minimal RNA-polymerase-III--specific promoter element from -49 to +17. The phosphocellulose 0.3-M-KCl fraction is absolutely required for transcription by RNA polymerase III, while the 0.85-M-KCl fraction represses aberrant transcription from incorrect initiation sites. Analysis of linker-substitution mutations indicated that the TATA-like sequence is required for RNA-polymerase-III--specific transcription. Although point mutations in the 5' half of the TATA-like sequence did not affect transcription, those in the 3' half shifted the transcription initiation site 3 bp upstream. The results suggest the the Na+/K(+)-ATPase alpha 1 subunit gene promoter contains a TATA-like sequence which can direct transcription by RNA polymerase III in vitro. The mechanism of alternative regulation of RNA polymerase II and RNA polymerase III is discussed.

Base Sequence↗
PubMed2003

Loss of the Rpb4/Rpb7 subcomplex in a mutant form of the Rpb6 subunit shared by RNA polymerases I, II, and III.

We have identified a conditional mutation in the shared Rpb6 subunit, assembled in RNA polymerases I, II, and III, that illuminated a new role that is independent of its assembly function. RNA polymerase II and III activities were significantly reduced in mutant cells before and after the shift to nonpermissive temperature. In contrast, RNA polymerase I was marginally affected. Although the Rpb6 mutant strain contained two mutations (P75S and Q100R), the majority of growth and transcription defects originated from substitution of an amino acid nearly identical in all eukaryotic counterparts as well as bacterial omega subunits (Q100R). Purification of mutant RNA polymerase II revealed that two subunits, Rpb4 and Rpb7, are selectively lost in mutant cells. Rpb4 and Rpb7 are present at substoichiometric levels, form a dissociable subcomplex, are required for RNA polymerase II activity at high temperatures, and have been implicated in the regulation of enzyme activity. Interaction experiments support a direct association between the Rpb6 and Rpb4 subunits, indicating that Rpb6 is one point of contact between the Rpb4/Rpb7 subcomplex and RNA polymerase II. The association of Rpb4/Rpb7 with Rpb6 suggests that analogous subunits of each RNA polymerase impart class-specific functions through a conserved core subunit.

Amino Acid Sequence↗
PubMed2003

Nucleotides from -16 to -12 determine specific promoter recognition by bacterial sigmaS-RNA polymerase.

The alternative sigma factor sigmaS, mainly active in stationary phase of growth, recognizes in vitro a -10 promoter sequence almost identical to the one for the main sigma factor, sigma70, thus raising the problem of how specific promoter recognition by sigmaS-RNA polymerase (EsigmaS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EsigmaS at the strictly sigmaS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB -10 sequence (-12C), instead of the T nucleotide canonical for sigma70-dependent promoters, is the major determinant for selective recognition by EsigmaS. The presence of the -12C does not allow formation of an open complex fully proficient in transcription initiation by Esigma70. The role of -12C as specific determinant for promoter recognition by EsigmaS was confirmed by sequence analysis of known EsigmaS-dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that EsigmaS, unlike Esigma70, can recognize both C and T as the first nucleotide in the -10 sequence. Additional promoter features such as the presence of a C nucleotide at position -13, contributing to open complex formation by EsigmaS, and a TG motif found at the unusual -16/-15 location, possibly contributing to initial binding to the promoter, also represent important factors for sigmaS-dependent transcription. We propose a new sequence, TG(N)0-2CCATA(c/a)T, as consensus -10 sequence for promoters exclusively recognized by EsigmaS.

Bacterial Proteins↗