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PubMed2004

Nucleotide sequence of RNA2 of Lettuce big-vein virus and evidence for a possible transcription termination/initiation strategy similar to that of rhabdoviruses.

Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210-1403 on the viral RNA), ORF2 (nt 1493-2494), ORF3 (nt 2617-3489), ORF4 (nt 3843-4337) and ORF5 (nt 4530-5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3' end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3'-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5'-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.

3' Flanking Region↗
PubMed1991

Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase.

Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.

Bacillus subtilis↗
PubMed2006

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription.

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

Algal Proteins↗
PubMed1988

RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA.

Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.

Base Sequence↗
PubMed1993

In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product.

The katF/rpoS gene product (sigma s), a central regulator of stationary-phase gene expression in Escherichia coli, has been purified from an overproducing strain. sigma s was used as an immunogen for the production of monoclonal antibodies. Previous sequence analysis of sigma s strongly indicated homology to the sigma factor family. We show biochemically in this paper that sigma s is a sigma factor. This protein can bind to core RNA polymerase (E), and this binding can be competed effectively by the major E. coli transcription initiation factor, sigma 70. Immunopurified sigma s holoenzyme (E sigma s) transcribes the promoters of the bolAp1 gene and the xthA gene. Interestingly, both promoters can also be transcribed by sigma 70 holoenzyme (E sigma 70).

Bacterial Proteins↗
PubMed1983

Characterization of the Escherichia coli gene for serine hydroxymethyltransferase.

Plasmid pGS1 carries the Escherichia coli glyA gene and its neighboring regions on a 13-kb EcoRI insert. In a cell-free transcription-translation system, the insert directs the synthesis of two polypeptides with Mr values of about 46 500 and 45 500. When the glyA gene is inactivated with the transposable element Tn5, the Mr 46 500 polypeptide is not observed, identifying it as the glyA gene product. The Mr 45 500 polypeptide is the product of an unknown gene designated gene X. When plasmids with random insertions of the Tn5 element in either the glyA gene or gene X are used as templates in the cell-free transcription-translation system, the polypeptides observed are smaller than the glyA or X gene products. A comparison of the site of each Tn5 insertion within the glyA gene or within gene X and the size of the polypeptide observed in the cell-free system enabled us to determine the direction of transcription and translation of both genes. The glyA gene is transcribed and translated in a direction opposite to that of gene X. Nucleotide sequencing confirmed the location and orientation of the two genes in the insert. DNase I footprinting experiments defined the glyA gene and gene X control regions recognized by RNA polymerase, and S1 nuclease mapping experiments located the transcription start point for each gene. The transcription start points for the two genes are 216 bp apart, and the translation start sites are 327 bp apart. Less than 90 bp separate the two RNA polymerase molecules bound to the two promoters.

Base Sequence↗
PubMed2004

Characterization of a rice nuclear-encoded plastid RNA polymerase gene OsRpoTp.

We isolated and characterized two rice genes, OsRpoTp and OsRpoTm, that encode putative phage-type RNA polymerases. Predicted amino acid sequences showed high homology of these genes to known RpoT genes. A transient expression assay using green fluorescent protein indicated that the encoded proteins were localized to plastids and mitochondria, respectively. We demonstrated by reverse transcription-PCR experiments and immunoblot analysis that OsRpoTp expression occurred at an early stage of leaf development, prior to the transcript accumulation of the genes that were transcribed by the nuclear-encoded plastid RNA polymerase (NEP). Expression analyses of the chloroplast-deficient rice mutant, virescent-1, showed a discrepancy between OsRpoTp protein accumulation and the level of transcripts of NEP-transcribed genes. Our results suggest that NEP activation is regulated by a process after transcription, and is affected by the developmental state of chloroplast biogenesis.

Base Sequence↗
PubMed1995

Requirements for activity of the yeast mitotic recombination hotspot HOT1: RNA polymerase I and multiple cis-acting sequences.

When inserted at novel locations in the yeast genome, the Saccharomyces cerevisiae recombination hotspot HOT1 stimulates mitotic exchange in adjacent sequences. HOT1 is derived from the rDNA repeat unit, and the sequences required for the recombination-stimulatory activity closely correspond to the rDNA transcription enhancer and initiation site, suggesting there is an association between high levels of RNA polymerase I transcription and increased recombination. To directly test whether RNA polymerase I is essential for HOT1 activity, a subunit of RNA polymerase I was deleted in a strain in which rRNA is transcribed by RNA polymerase II. HOT1 is completely inactive in this strain. Deletion analysis and site-directed mutagenesis were used to further define the sequences within the rDNA enhancer required for HOT1 activity. These studies show that the enhancer contains at least four distinct regions that are required for hotspot activity. In most cases mutations in these regions also decrease transcription from this element, further confirming the association of recombination and transcription.

Base Sequence↗
PubMed2001

Identification and characterization of cis elements in the STAT3 gene regulating STAT3 alpha and STAT3 beta messenger RNA splicing.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulator of multiple cell-fate decisions, including myeloid cell differentiation. Two isoforms of STAT3 have been identified: alpha (p92) and beta (p83). These differ structurally in their C-terminal transactivation domains, resulting in distinct functional activities. The cis genetic elements that regulate the ratio of alpha to beta messenger RNA (mRNA) are unknown. In this study, cloning, sequencing, and splicing analysis of the human and murine STAT3 genes revealed a highly conserved 5' donor site for generation of both alpha and beta mRNA and distinct branch-point sequences, polypyrimidine tracts, and 3' acceptor sites (ASs) for each. The beta 3' AS was found to be located 50 nucleotides downstream of the alpha 3' AS in exon 23. Two additional cryptic 3' ASs (delta and epsilon) were also identified. Thus, we identified for the first time the cis regulatory sequences responsible for generation of STAT3 alpha and STAT3 beta mRNA.

Animals↗
PubMed2006

Transient state kinetics of transcription elongation by T7 RNA polymerase.

The single subunit DNA-dependent RNA polymerase (RNAP) from bacteriophage T7 catalyzes both promoter-dependent transcription initiation and promoter-independent elongation. Using a promoter-free substrate, we have dissected the kinetic pathway of single nucleotide incorporation during elongation. We show that T7 RNAP undergoes a slow conformational change (0.01-0.03 s(-1)) to form an elongation competent complex with the promoter-free substrate (dissociation constant (Kd) of 96 nM). The complex binds to a correct NTP (Kd of 80 microM) and incorporates the nucleoside monophosphate (NMP) into RNA primer very efficiently (220 s(-1) at 25 degrees C). An overall free energy change (-5.5 kcal/mol) and internal free energy change (-3.7 kcal/mol) of single NMP incorporation was calculated from the measured equilibrium constants. In the presence of inorganic pyrophosphate (PPi), the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 s(-1) with PPi Kd of 1.2 mM. Several experiments were designed to investigate the rate-limiting step in the pathway of single nucleotide addition. Acid-quench and pulse-chase kinetics indicated that an isomerization step before chemistry is rate-limiting. The very similar rate constants of sequential incorporation of two nucleotides indicated that the steps after chemistry are fast. Based on available data, we propose that the preinsertion to insertion isomerization of NTP observed in the crystallographic studies of T7 RNAP is a likely candidate for the rate-limiting step. The studies here provide a kinetic framework to investigate structure-function and fidelity of RNA synthesis and to further explore the role of the conformational change in nucleotide selection during RNA synthesis.

Bacteriophage T7↗
PubMed1983

RNAs from two independently isolated defective interfering particles of Sindbis virus contain a cellular tRNA sequence at their 5' ends.

Defective interfering (DI) particles are deletion mutants that interfere specifically with the replication of homologous standard virus. We have determined the 5'-terminal nucleotide sequences of two DI RNA populations by the following methods: (i) cloning of the cDNA from one of the DI RNA populations and sequencing a representative clone, and (ii) using both DI RNA populations as templates for preparing primer-directed cDNA transcripts and sequencing these transcripts. The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp.

Animals↗
PubMed1993

Active center rearrangement in RNA polymerase initiation complex.

His1237 in the beta subunit of Escherichia coli RNA polymerase marks the "5' face" of the active center since it can be cross-linked to the gamma-phosphate of the priming substrate. It is demonstrated that RNA chains up to 9 nucleotides in length can be synthesized using His1237-cross-linked nucleotide as a primer. Thus, a substantial mass of RNA can be accommodated in the active center between His1237 and the site of catalysis that remains juxtaposed to the growing 3' end. The apparent "filling" of the active center with RNA precedes promoter clearance and suggests a mechanism of coupling between catalysis and saltatory translocation of RNA polymerase.

Amino Acid Sequence↗
PubMed1998

Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3Dpol with VPg and with genetic variants of 3AB.

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

Amino Acid Sequence↗
PubMed1992

Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.

The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.

Animals↗
PubMed1999

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity.

Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position -1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the alpha subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Amino Acid Sequence↗
PubMedDate not supplied

RpoA: a useful gene for phylogenetic analysis in diatoms.

The aim of this study was to compare the usefulness of two chloroplast-encoded genes (rpoA and rbcL) and the nuclear-encoded small subunit (SSU) ribosomal RNA for reconstructing phylogenetic relationships among diatoms at lower taxonomic levels. To this end, the rpoA and rbcL genes for selected centric and pennate diatoms were sequenced. The new rpoA and rbcL sequences, and an existing nuclear-encoded SSU rRNA data set, were subjected to weighted/unweighted parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. All of the tree-building methods employed showed, based on the support values, that the rpoA gene was the most useful, relative to the rbcL and SSU rRNA genes, in determining phylogenetic relationships among the sampled diatoms. The support values for the relationships among the pennate lineages were, in many instances, greater in the rpoA trees than in the SSU rRNA trees. These results suggest that rpoA might be of value in determining phylogenetic relationships among pennate lineages.

Base Sequence↗
PubMed2000

Comparison of identical temperature-sensitive mutations in the L polymerase proteins of sendai and parainfluenza3 viruses.

The L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal.

Amino Acid Sequence↗
PubMed1994

Human RNA polymerase II subunit hRPB14 is homologous to yeast RNA polymerase I, II, and III subunits (AC19 and RPB11) and is similar to a portion of the bacterial RNA polymerase alpha subunit.

The cDNA cloning of the human polII 14-kDa subunit, hRPB14, and the comparison of its aa sequence with those of other pol subunits are described. The aa sequence of hRPB14 has homology to yeast poIII subunit RPB11 (44%), to a common subunit of yeast polI and polIII AC19 (24%) and to a Caenorhabditis elegans sequence (33%). hRPB14 contains a 19-aa motif, located in its N terminus, which was also found in human polII 33-kDa subunit hRPB33, yeast pol subunits (AC40, AC19, RPB3 and RPB11), and in the bacterial pol alpha subunit, which was involved in subunit assembly. This motif was also conserved in the conjugation-specific gene products of Tetrahymena (CnjC), Merchantia polymorpha chloroplast DNA (RNLVA) and C. elegans DNA (CEF58A4; deduced from the nucleotide sequence and of unknown function). The evolutionary emergence of a probable eukaryotic heterodimer, hRPB14/hRPB33, from a prokaryotic homodimer, alpha 2, is hypothesized.

Amino Acid Sequence↗