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At least 541 records · Page 30Linked to original sources
PubMed1994

Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB.

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA polymerase 3Dpol (280 ng/ml) is strongly stimulated (50-100 fold) by the addition of purified poliovirus polypeptide 3AB. The synthesis of product continues linearly with time for up to 90 min. The reaction with 3Dpol alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Optimal stimulation is achieved under conditions where the concentration of 3Dpol and of template is low, when the molar ratio of 3AB to 3Dpol is about 100:1 and that of 3AB to poly(A) is about 25:1. In the presence of 3AB, the yield of product made by 3Dpol is much increased but its size is unchanged. From a number of basic proteins and peptides tested, a few were found which also exhibited limited enhancement of polymerase activity. The stimulatory effect of 3AB is probably related to its ability to bind both the template-primer, poly(A).oligo(dT)15, and 3Dpol (Molla, A., Harris, K. S., Paul, A. V., Shin, S. H., Mugavero, J., and Wimmer, E. J. (1994) J. Biol. Chem. 269, 27015-27020). RNA synthesis on purified poliovirus RNA with oligo(dT)15 primer is enhanced by 3AB about 5-10 fold, and this reaction is highly sensitive to detergent.

Amino Acid Sequence↗
PubMed1996

A branched pathway in the early stage of transcription by Escherichia coli RNA polymerase.

The time-course of synthesis of long and short (abortive) transcripts by Escherichia coli RNA polymerase was investigated under single round conditions in vitro. The synthesis of long RNA initiated at the lambda PR (with an altered leader sequence) or at the lacUV5 promoter was completed within five minutes, but abortive transcripts were continuously synthesized for at least 20 minutes. The results indicate the presence of transcription complexes that are capable only of abortive synthesis, and not of productive elongation. Consistently, only one in four of the RNA polymerase molecules that initially associated with the lambda PR promoter synthesized long RNA. The enzyme reisolated from productive complexes synthesized both long and abortive transcripts, behaving just like the original enzyme. This suggests that RNA polymerase is homogeneous with respect to its ability to synthesize the two types of product. Overall, the results indicate that many transcription complexes can be irreversibly trapped in vitro in an abortive cycle.

Base Sequence↗
PubMed1998

Protein-primed RNA synthesis by purified poliovirus RNA polymerase.

A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus genomic RNA. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5' end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5' mRNA end, has solved the first conundrum. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5'-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U). Initiation of transcription of the poliovirus genome from the polyadenylated 3' end therefore depends on VPg.

Amino Acid Sequence↗
PubMed1993

The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling.

Most Escherichia coli promoters studied so far form stable open complexes with sigma 70-RNA polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge. A few exceptions are nevertheless known. The analysis of a number of promoters in Bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma A-RNA polymerase may be a more general phenomenon than in Escherichia coli. We show that the main early and late promoters from the Bacillus subtilis phage phi 29 form unstable open complexes that are stabilized either by the formation of the first phosphodiester bond between the initiating nucleoside triphosphates or by DNA supercoiling. The functional characteristics of these two strong promoters suggest that they are not optimized for a tight and stable RNA polymerase binding. Their high activity is probably the consequence of the efficiency of further steps leading to the formation of an elongation complex.

Bacillus Phages↗
PubMed1988

3D gene of foot-and-mouth disease virus. Conservation by convergence of average sequences.

The nucleotide sequence of the 3D (polymerase) gene of eight epidemiologically related isolates of foot-and-mouth disease virus of serotype C1 is reported. The genetic heterogeneity of 3D RNA is compared with that of the VP1-coding RNA of the same viruses. Regression lines of substitutions per nucleotide that distinguish any pair of viruses as a function of the time interval between the corresponding isolations show: (1) the slope (substitutions/nucleotide per month) is 2.1 times larger for the VP1 RNA than for the 3D RNA region; (2) the intercept with the ordinate (substitutions/nucleotide) for VP1 RNA is indistinguishable from that for 3D RNA. Thus, the average heterogeneity of the VP1-coding region is very similar to that of the 3D-coding region only among co-circulating viruses. Nine mutations and points of heterogeneity occurred within nucleotide residues 883 to 1026, which encode an amino acid segment, extremely conserved among many different RNA viruses. The results suggest that, rather than due to inherently lower mutability, the conservation of 3D genes is caused by a limitation in the fixation of substitutions in viable genomes.

Base Sequence↗
PubMed1990

Vesicular stomatitis virion-associated transcriptase activity was suppressed in vitro by a synthetic 21 amino acid oligopeptide prepared to mimic the carboxy-terminus of NS protein.

To study the biological function of the NS protein of vesicular stomatitis virus (VSV), we prepared 21 species of synthetic oligopeptides with 11-21 amino acid residues, corresponding to every portion of the amino acid sequence of NS protein (Indiana serotype), and tested their effects on the VS virion (VSV) transcriptase activity in vitro. Only one peptide affected the virion-associated transcriptase activity of VSV Indiana, by reducing the incorporation of [3H]GMP into acid-insoluble fraction (IC50 = 26 microM). This peptide, the amino acid sequence of which corresponded to the carboxy (C)-terminal region of NS protein, also inhibited the New Jersey serotype virus transcriptase activity, as expected from a high degree of homology found between the amino acid sequences of the C-terminal regions of NS protein of both serotype viruses. Electrophoretic analysis on acrylamide gels of RNA transcripts revealed that the inhibitory synthetic peptide decreased the frequency of the initiation of transcription with no apparent effect on the chain-elongation process of viral transcription. As expected from its highly conserved amino acid sequence, these results suggest that the C-terminal domain of VSV NS protein is involved in initiating viral RNA synthesis.

Amino Acid Sequence↗
PubMed1998

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro.

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Acylation↗
PubMed1994

A non-essential domain of Escherichia coli RNA polymerase required for the action of the termination factor Alc.

An evolutionarily nonconserved region of approximately 250 amino acids can be deleted from the amino-terminal part of the beta subunit of Escherichia coli RNA polymerase without effect on the enzyme's basic function. The non-essential segment is located between two highly conserved motifs and is flanked by sequences participating in the rifampicin-binding site. The results define the second non-essential domain in the beta subunit, in addition to the more distal dispensable segment identified previously. The Alc protein of bacteriophage T4 participates in the host transcription shutoff after infection by causing premature termination of transcription on E. coli DNA. Point mutations which prevent Alc action in vivo change amino acids in the non-essential NH2-terminal domain of the beta subunit. These point mutations as well as deletions which remove the non-essential region also prevent Alc action. Thus, in the RNA polymerase molecule, the proximal non-essential domain of beta may function as an acceptor of Alc or other regulatory factors.

Amino Acid Sequence↗
PubMed1994

A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA.

The genome of all retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. We report the results of experiments designed to identify the 5' and 3' boundaries of the dimerization domain of the HIV-1 genome: (1) An HIV-1 RNA starting at nucleotide 252 or at other downstream positions (four tested) does not dimerize despite the inclusion of the whole of a previously proposed dimerization domain (nucleotides 295-401); (2) an RNA starting between nucleotides 242 and 249 (five positions tested) dimerizes to a variable extent depending on the starting position; (3) an RNA starting at nucleotide 233 or at other upstream positions (five tested) is fully or > 80% dimeric; (4) an RNA starting at nucleotide 1 but lacking the 233-251 or the 242-251 region is, respectively, fully monomeric or about 50% monomeric; (5) the 343-401 region contains two strings of G's (GGGGG367 and GGG384) that had been postulated to promote genome dimerization through the formation of guanine quartets. We have deleted the 379-401, 358-401, and 343-401 regions from otherwise dimeric RNAs without changing their ability to dimerize. We reach three conclusions: (1) a dimerization signal exists upstream of the major 5' splice donor (nucleotide 290); (2) the previously proposed downstream dimerization domain is insufficient to promote dimerization and has a 3' half that is not necessary to obtain fully dimeric RNAs; (3) the 5' boundary of the HIV-1 dimerization domain is located somewhere between nucleotides 233 and 242, and the 3' boundary is located no farther than at nucleotide 342, making it possible that the 5' and 3' boundaries of the HIV-1 dimerization domain are both located within the leader sequence. We speculate that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA.

Base Sequence↗
PubMed1990

RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage.

We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [alpha-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.

Base Sequence↗
PubMed1990

Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.

The OmpR binding sequence (OBS) in the upstream region of the ompF promoter of Escherichia coli was fused to 27 synthetic promoters. Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator. In vivo DNA footprinting revealed that OmpR enhanced the binding of RNA polymerase to the promoters. This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription. These results indicate that OmpR stabilizes the formation of an RNA polymerase-promoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression.

Bacterial Outer Membrane Proteins↗
PubMed1993

Enzymatic recognition of the base pair between isocytidine and isoguanosine.

The ability of various polymerases to catalyze the template-directed formation of a base pair between isoguanine (iso-G) and isocytosine (iso-C) in duplex oligonucleotides has been investigated. A new procedure was developed for preparing derivatives of deoxyisoguanosine suitable for incorporation into DNA using an automated DNA synthesizer. T7 RNA polymerase, AMV reverse transcriptase, and the Klenow fragment of DNA polymerase all incorporated iso-G opposite iso-C in a template. T4 DNA polymerase did not. Several polymerases also incorporated iso-G opposite T, presumably through pairing with a minor tautomeric form of iso-G complementary to T. In a template, iso-G directs the incorporation of both iso-C and T when Klenow fragment is the catalyst and only U when T7 RNA polymerase is the catalyst. Further, derivatives of iso-C were found to undergo significant amounts of deamination under alkaline conditions used for base deprotection after automated oligonucleotide synthesis. Both the deamination reaction of iso-C and the ambivalent tautomeric forms of iso-G make it unlikely that the (iso-C).(iso-G) base pair was a part of information storage molecules also containing the A.T and G.C base pairs found in primitive forms of life that emerged on planet earth several billion years ago. Nevertheless, the extra letters in the genetic alphabet can serve useful roles in a contemporary laboratory setting.

Adenosine↗
PubMed1989

Promoter analysis of influenza virus RNA polymerase.

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.

Base Sequence↗
PubMed1996

Plasmid RNA polymerase-like mitochondrial sequences in Agaricus bitorquis.

A linear mitochondrial plasmid, pEM, found in certain isolates of the basidiomycete Agaricus bitorquis, potentially encodes virus-like DNA and RNA polymerases. Mitochondrial DNA from Agaricus bisporus that hybridizes to an internal region of pEM contains a fragmented and potentially non-functional version of the carboxy terminal end of the plasmid RNA polymerase. In this study, we present the sequence of the corresponding region of mitochondrial DNA from A. bitorquis. This sequence contained the same region of the plasmid RNA polymerase gene as was reported for the mitochondrial DNA of A. bisporus, and the level of similarity between the A. bisporus and A. bitorquis mitochondrial sequences was much higher than the level of similarity between either mitochondrial sequence and the plasmid. We propose that this plasmid RNA polymerase-like sequence was present in the Agaricus mitochondrial genome before the divergence of A. bisporus and A. bitorquis, and thus is unlikely to be a recent derivative of the plasmid pEM.

Agaricus↗
PubMed1993

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.

Animals↗
PubMed2004

The bipartite 3'-cis-acting signal for replication is required for formation of a ribonucleoprotein complex in vivo between the viral genome and its RNA polymerase in yeast 23 S RNA virus.

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.9 kb) encodes only its RNA-dependent RNA polymerase, p104, and forms a ribonucleoprotein complex with p104 in vivo. Previously we succeeded in generating 23 S RNA virus in yeast from an expression vector containing the entire viral cDNA sequence. Using this system, we have recently identified a bipartite 3' cis-acting signal for replication. The signal consists of a stretch of four cytidines (Cs) at the 3' end and a mismatched pair of purines in a stem-loop structure that partially overlaps the terminal four Cs. Although the 3' terminal and penultimate Cs are not essential for virus launching, the generated viruses efficiently recovered these terminal nucleotides. In this work, we expressed RNA transcripts containing the entire 23 S RNA genome but incapable of generating the virus because of the presence of non-viral extra sequences at the 3' ends. These transcripts could form complexes with p104 in vivo, and a detailed analysis indicated that the mismatched pair of purines as well as the third and fourth Cs from the viral 3' end was essential for this complex-forming activity. Given that 23 S RNA virus does not have genes for capsid proteins, the binding of p104 to the viral 3' end, in addition to the efficient 3' terminal repair, may play a crucial role in virus persistence by protecting and maintaining the correct viral 3' end in vivo.

Base Sequence↗
PubMed1991

RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay.

We have used a self-cleaving RNA molecule (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates. When the nascent transcript attains the minimum length required for the "hammerhead" domain of the transcript to fully emerge from the ternary complex, the "hammerhead" structure forms and self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to that generated by using a noncleaving control template. We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.

Base Sequence↗
PubMed1995

In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle.

An in vitro transcription assay was used to study transcription from synthetic RNA corresponding to the 3' terminus of influenza A virus cRNA. Micrococcal nuclease-treated influenza virus ribonucleoprotein was used as a source of active polymerase complex. Mutations at two regions of the 13 nucleotide-long conserved cRNA 3' terminus were shown to reduce transcription templated by the short added model RNAs. The first region, at positions 1 and 2 from the 3' terminus, was shown to be affected by the exact nature of the dinucleotide primer used in the in vitro transcription reactions and may not be relevant in vivo. The second region, centred on positions 11 and 12, may be involved in base pairing with conserved nucleotides at the 5' terminus of the cRNA. Evidence for this comes from the finding that RNA corresponding to 5' conserved sequences, but mutated to restore the postulated base pairing with the mutated 3' ends, could partly restore transcription. Binding of the influenza virus polymerase complex to a set of 5'-mutated RNAs was investigated using a photochemical cross-linking assay. Specific binding to two regions of the cRNA 5' terminus was demonstrated, at positions 1 to 3 and positions 8 to 10. Together, these observations suggest that a panhandle forms from the termini of the cRNA molecule and that this structure may play a role in transcription to produce virion RNA.

Base Sequence↗