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PubMed1990

ret1-1, a yeast mutant affecting transcription termination by RNA polymerase III.

In eukaryotes, extended tracts of T residues are known to signal the termination of RNA polymerase III transcription. However, it is not understood how the transcription complex interacts with this signal. We have developed a selection system in yeast that uses ochre suppressors weakened by altered transcription termination signals to identify mutations in the proteins involved in termination of transcription by RNA polymerase III. Over 7600 suppression-plus yeast mutants were selected and screened, leading to the identification of one whose effect is mediated transcriptionally. The ret1-1 mutation arose in conjunction with multiple rare events, including uninduced sporulation, gene amplification, and mutation. In vitro transcription extracts from ret1-1 cells terminate less efficiently at weak transcription termination signals than those from RET1 cells, using a variety of tRNA templates. In vivo this reduced termination efficiency can lead to either an increase or a further decrease in suppressor strength, depending on the location of the altered termination signal present in the suppressor tRNA gene. Fractionation of in vitro transcription extracts and purification of RNA polymerase III has shown that the mutant effect is mediated by highly purified polymerase in a reconstituted system.

Alleles↗
PubMed1994

Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis.

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass, M. G. Mulkerrin, and J. A. Wells, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paoni, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. Zoller, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragenic suppression analysis of such mutant viruses may be very useful in identifying components of the viral replication complex.

Alanine↗
PubMed1999

Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA.

cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.

Alfalfa mosaic virus↗
PubMed1993

Molecular phylogeny of Drosophila based on ribosomal RNA sequences.

Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.

Animals↗
PubMedDate not supplied

Multiple alignment and hierarchical clustering of conserved amino acid sequences in the replication-associated proteins of plant RNA viruses.

We have used multiple alignment computer programs to align and hierarchically cluster the conserved amino acid "signature" sequences found in the replication-associated proteins of all plant RNA viruses sequenced so far. These regions, called "polymerase", "nucleotide-binding" and "N-terminal" are well conserved even between viruses which are only distantly related, and are thus very well suited for this type of analysis. Our results show that the clusterings obtained using these very short amino acid sequences are very robust to computing parameters and are surprisingly well matched with the taxonomic grouping of RNA plant viruses. The possibility of using this system as a new taxonomic criterion is discussed.

Amino Acid Sequence↗
PubMed1992

Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Base Sequence↗
PubMed1994

Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription.

Hepadnavirus DNA minus strands are covalently linked at their 5' terminus to the viral P gene product, which has been taken to indicate that the hepadnaviral polymerase polypeptide itself also functions as a protein primer for initiating reverse transcription of the RNA pregenome. The present study confirms this indication by identifying the nucleotide-linked amino acid in the P protein sequence of the duck hepatitis B virus (DHBV). In a first set of experiments, mutational analysis of three phylogenetically conserved tyrosine residues in the DNA terminal (TP) domain indicated that of these, only tyrosine 96 was essential for both viral DNA synthesis in transfected cells and priming of DNA synthesis in a cell-free system. This assignment was confirmed by direct biochemical analysis: tryptic peptides from the DHBV P protein, 32P labelled at the priming amino acid by the initiating dGTP and additionally labelled internally by [35S]methionine, were isolated and analyzed in parallel to reference peptides synthesized chemically and 33P labelled by a tyrosine kinase. Mobility in high-performance liquid chromatography, as well as the release in stepwise amino acid sequencing of phospholabel and of [35S]methionine, identified the priming amino acid unequivocally as the tyrosine in the sequence 91KLSGLYQMK99, which is located in the center of the TP domain. Conserved sequence motifs surrounding Tyr-96 allow the prediction of the priming tyrosine in other hepadnaviruses. Weak sequence similarity to picornavirus genome-linked polypeptides (VPgs) and similar gene organization suggest a common origin for the mechanisms that use protein priming to initiate synthesis of viral DNA genomes or RNA genomes from an RNA template.

Amino Acid Sequence↗
PubMed1998

The late expression factors 8 and 9 and possibly the phosphoprotein p78/83 of Autographa californica multicapsid nucleopolyhedrovirus are components of the virus-induced RNA polymerase.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Amino Acid Sequence↗
PubMed1994

Factor-dependent transcription termination by vaccinia RNA polymerase. Kinetic coupling and requirement for ATP hydrolysis.

Transcription termination during synthesis of vaccinia early mRNAs occurs downstream of a UUUUUNU signal in the nascent transcript and requires a virus-encoded termination factor (VTF), which is identical with the vaccinia mRNA capping enzyme. Using purified transcription complexes halted at defined sites on linear DNA templates, we have examined the order and timing of events during a single round of elongation and termination. We find that although cap synthesis occurs by the time the nascent RNA is 31 nucleotides long, capping enzyme is not stably associated with the elongation complex at this stage. Stable interaction, defined by the formation of a termination-competent complex, requires a longer nascent RNA, e.g. 51 nucleotides, but does not depend on prior transcription of the termination signal. The acquisition of termination competence correlates temporally with the physical association of capping enzyme/VTF with the elongation complex, as revealed by UV cross-linking of the capping enzyme large subunit to the nascent RNA chain. Subsequent induction of termination and transcript release by capping enzyme requires energy, specifically the hydrolysis of ATP. The choice of termination site is flexible and is determined by a kinetic balance between the rate of polymerase elongation and the rate of signaling. Signaling rate is related directly to the concentration of hydrolyzable ATP. An apparent lower limit of 33 nucleotides between the 5' boundary of the termination signal and the most proximal termination site implies that the UUUUUNU signal must be extruded from the RNA polymerase before it can be acted upon by VTF. Similarities between VTF-dependent termination and rho-dependent termination underscore an evolutionarily conserved mechanism for RNA signal transduction to the elongating RNA polymerase.

Adenosine Triphosphate↗
PubMed1994

Sequence analysis and compositional properties of untranslated regions of human mRNAs.

A detailed computer analysis of the untranslated regions, 5'-UTR and 3'-UTR, of human mRNA sequences is reported. The compositional properties of these regions, compared with those of the corresponding coding regions, indicate that 5'-UTR and 3'-UTR are less affected by the isochore compartmentalization than the corresponding third codon positions of mRNAs. The presence of higher functional constraints in 5'-UTR is also reported. Dinucleotide analysis shows a depletion of CpG and TpA in both sequences. A search for significant sequence motifs using the WORDUP algorithm reveals the patterns already known to have a functional role in the mRNA UTR, and several other motifs whose functional roles remain to be demonstrated. This type of analysis may be particularly useful for guiding site-directed mutagenesis experiments. In addition, it can be used for assessing the nature of anonymous sequences now produced in large amounts in megabase sequencing projects.

Algorithms↗
PubMed1989

T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide.

The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production of the five-base runoff transcript GGACU. In particular, Km for the interaction between enzyme and template under saturating levels of all four nucleotide triphosphates was shown to be approximately 0.02 microM. We now show that the corresponding Km and Vmax for initiation on a similar template coding for the runoff transcript GACU are the same as for the earlier study (Km = 0.02 microM; kcat = 40-50 min-1). This new template allows the measurement Km for association of the initial nucleotide GTP with enzyme or with the enzyme-DNA complex. The results show that KGTPm (0.60 mM) is somewhat higher than earlier approximations of Km for addition of elongating GTP during the later phase of processive elongation. As expected, the (initiating) Km for the GTP analogue ITP (KITPm) is increased (by about 2-fold), presumably as a result of weakened Watson-Crick base pairing. However, comparison of Km values for the GTP analogues GMP and guanosine shows little effect on substitution of the 5'-triphosphate by monophosphate or by a hydroxyl, respectively. This result suggests that a single active site has been evolutionarily adapted to accept from the 5' end of a waiting nucleotide both a 5'-triphosphate at initiation and a 5'-monophosphate ester (RNA) during elongation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1996

Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

Base Sequence↗
PubMed1994

GreA-induced transcript cleavage is accompanied by reverse translocation to a different transcription complex conformation.

GreA- and GreB-induced transcript cleavage drives reverse translocation of Escherichia coli RNA polymerase on a DNA template in the absence of NTPs (Feng, G.-H., Lee, D. N., Wang, D., Chan, C. L., and Landick, R. (1994) J. Biol. Chem. 269, 22282-22294, accompanying report). During transcript elongation, the sizes of the DNA footprint and the single-stranded transcription bubble vary markedly among transcription complexes halted at different template positions. To test whether transcription complex intermediates formed during transcript cleavage-induced reverse translocation also display heterogeneous conformations at different template positions, we examined the structures of two different transcription complexes before and after GreA treatment. Transcription complexes halted at position +16 after initiation at the T7 A1 promoter or paused at the trpL pause site exhibited strong blocks to transcript cleavage after removal of 6 to 10 nucleotides. In both cases, the down-stream contact between RNA polymerase and DNA moved little during transcript cleavage, thereby increasing its distance from the active site, whereas the upstream DNA contact and the borders of the transcription bubble moved in approximate register with the transcript 3'-end. The backward movements of halted E. coli RNA polymerase are similar to a recently postulated model for discontinuous translocation during transcription, but differ from those reported for arrested RNA polymerase II transcription complexes.

Bacterial Proteins↗
PubMed1991

Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe.

The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected.

Base Sequence↗
PubMed2004

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR.

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.

Amino Acid Sequence↗
PubMed1990

A sequence that directs transcriptional initiation in yeast.

While RNA polymerase II of the yeast Saccharomyces cerevisiae initiates transcription at discrete sites, these sites are located over a wide range of distances from the TATA box for different genes. This variability has led to a number of proposals for consensus sequences located at the initiation site which, in conjunction with the TATA box, would direct initiation. We tested this hypothesis via oligonucleotide-directed mutagenesis, by placing the sequence CAAG, a member of one of these consensus sequences, upstream of the coding sequence of the CYC7 gene at a site at which initiation does not occur. The distance between the TATA sequence and this putative initiation site was varied by inserting it into the wild-type gene and three deletion mutants. The results demonstrated that this sequence can serve as an initiation site when located 49, 77, or 106 bp from the TATA sequence, but not when located 30 bp away.

Base Sequence↗
PubMed2001

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding.

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

Adenosine Monophosphate↗