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At least 91 records · Page 5Linked to original sources

PubMed1997

Incompatibility of Escherichia coli rho mutants with plasmids is mediated by plasmid-specific transcription.

The Escherichia coli rho-15 mutant (deficient in transcription termination) is known to be incompatible with pBR322 and other plasmids (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We show that failure of pBR322 to transform rho-15 is mediated by transcription from the tet promoter and readthrough from the tet gene into the rom region. Using an isopropyl-beta-D-thiogalactopyranoside-inducible promoter to replace the tet promoter, we have demonstrated that plasmid-specific transcription inhibits growth of the rho-15 host, possibly due to the expression of the Rom protein. The involvement of Rom protein in pBR322-rho-15 incompatibility is further indicated by the following two experiments. (i) Functional inactivation of the rom gene in pBR322 enabled plasmids to transform E. coli rho-15. (ii) Specific overexpression of the rom gene abolished plasmid transformation into E. coli rho-15. An rpoB8(Ts) mutant RNA polymerase which compensated for the termination defect in E. coli rho-15 also restored plasmid-host compatibility, suggesting that Rom-mediated plasmid-host incompatibility is linked to a defect in transcription termination.

Bacterial Proteins↗

PubMed2006

In Planta production of immunogenic poliovirus peptide using tobacco mosaic virus-based vector system.

The tobacco mosaic virus (TMV) provides an attractive means of producing foreign peptides in plants. In this study, a TMV-based vector was designed such that a fragment encoding 15 amino acids of the poliovirus peptide (PVP) derived from the viral capsid proteins VP3 and VP1 of poliovirus type 1 Sabin was inserted downstream of the six-base 3' context nucleotide sequence of the TMV coat protein (CP) gene. This design allowed readthrough at the amber stop codon, thereby producing the chimeric TMV particle with both intact CP and CP-fusion protein (CP-PVP) in Nicotiana tabacum cv. Samsun infected with the TMV vector. The TMVCP-PVP virus particle induced antibodies against PVP as well as TMVCP in mice after intraperitoneal immunization. These data illustrate the potential of the readthrough translation system with TMVCP for antigen presentation and vaccine production.

Animals↗

PubMed1991

cis Acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus.

The pol gene of the Moloney murine leukemia virus (M-MuLV) is expressed as a Gag-Pol fusion protein through an in-frame suppression of the UAG termination codon located between the two genes. The role of nucleotide context in suppression was investigated, in a rabbit reticulocyte lysate translation system, using site-directed mutagenesis. The results indicate that the translational readthrough is mediated by at least 50 bases long RNA sequence located 3' to the gag UAG termination codon. Within this sequence a short purine-rich sequence adjacent to the amber codon, highly conserved among different retroviruses, appears essential for M-MuLV suppression. Two alternative putative stem and loop like RNA structures can be drawn at the gag-pol junction, one abutting the gag UAG codon, and the second downstream to it. None of these structures appears to be important to the suppression process.

Animals↗

PubMed1987

Transcription of the histone loci on lampbrush chromosomes of the newt Notophthalmus viridescens.

We have investigated transcription of the histone gene cluster on lampbrush chromosomes of the newt Notophthalmus viridescens. Clusters of the five histone genes are separated by long tracts of a DNA repeat called satellite 1. Previous in situ hybridization results demonstrated the presence of histone gene coding regions, intergene spacers, and flanking satellite repeats in giant transcription units on lampbrush chromosomes. These results suggested a model in which transcription initiates at histone gene promoters and continues through the rest of the gene cluster into satellite repeats. The readthrough transcription model predicts that spacer regions upstream of the first promoter in the gene cluster should be absent from nascent transcripts on chromosome loops. We have used such upstream spacer probes for in situ hybridization to lampbrush chromosomes. Contrary to our expectation, the results show that upstream spacer regions are transcribed, and indicate that transcription often initiates upstream of the first histone gene promoter. The relationship of giant transcription units in oocyte nuclei to cytoplasmic histone mRNA is discussed.

Animals↗

PubMed1998

Molecular basis for naturally occurring elevated readthrough transcription across the M-F junction of the paramyxovirus SV5.

Transcription of the paramyxovirus RNA genome is thought to involve a sequential stop-start mechanism whereby monocistronic mRNAs are produced by polyadenylation and termination of 3' upstream gene followed by reinitiation at the downstream start site. For a number of paramyxoviruses, transcription across the M-F gene junction results in the synthesis of high levels of a dicistronic M-F readthrough RNA. In cells infected with the paramyxovirus SV5, 15% or less of the transcripts from the viral P, M, SH, HN, and L genes were detected as readthrough products with the 3' proximal gene. By contrast, approximately 40% of the SV5 F mRNA was detected as a dicistronic M-F transcript. A comparison of the individual SV5 gene junctions showed that elevated M-F readthrough transcription correlate with the M gene end having the shortest U tract for directing polyadenylation and a gene end sequence that differs from the consensus sequence. We have tested the hypothesis that elevated M-F readthrough transcription results from an inefficient termination signal at the end of the M gene. A reverse genetics system was established whereby SV5 transcription was reconstituted in transfected cells using cDNA-derived polymerase components and dicistronic minigenomes that encoded either the SV5 M-F or the SH-HN gene junction. Chimeric SV5 minigenomes were constructed to contain exchanges of a 10 base gene end sequence and the U tract from the M-F (approximately 40% readthrough) and SH-HN (approximately 15% readthrough) junctions. Northern blot analysis of RNA synthesized from these altered templates showed that, in the context of the M-F intergenic region, increasing the length of the M gene end U tract from four residues to six or eight U residues did not decrease M-F readthrough transcription. In contrast, chimeric minigenomes that contained the 10 base region from the end of the SH gene directed very efficient gene termination and a corresponding decrease in readthrough transcription. Mutational analysis showed that a single G to A substitution located five bases 3' to the M gene U tract was sufficient to convert the M gene end region to an efficient signal for polyadenylation-termination. These results demonstrate a role for the gene end region located immediately 3' to the U tract as a major determinant of transcription termination in the paramyxovirus genome. The possible role of M-F readthrough transcription in the paramyxovirus growth cycle is discussed.

Animals↗

PubMed2005

The cotranscriptional assembly of snoRNPs controls the biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae.

The carboxy-terminal domain (CTD) of RNA polymerase II large subunit acts as a platform to assemble the RNA processing machinery in a controlled way throughout the transcription cycle. In yeast, recent findings revealed a physical connection between phospho-CTD, generated by the Ctk1p kinase, and protein factors having a function in small nucleolar RNA (snoRNA) biogenesis. The snoRNAs represent a large family of polymerase II noncoding transcripts that are associated with highly conserved polypeptides to form stable ribonucleoprotein particles (snoRNPs). In this work, we have studied the biogenesis of the snoRNPs belonging to the box H/ACA class. We report that the assembly factor Naf1p and the core components Cbf5p and Nhp2p are recruited on H/ACA snoRNA genes very early during transcription. We also show that the cotranscriptional recruitment of Naf1p and Cbf5p is Ctk1p dependent and that Ctk1p and Cbf5p are required for preventing the readthrough into the snoRNA downstream genes. All these data suggest that proper cotranscriptional snoRNP assembly controls 3'-end formation of snoRNAs and, consequently, the release of a functional particle.

Chromatin Immunoprecipitation↗

PubMed1997

Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes.

Efficiency of translation termination is affected if the last two amino acids in the nascent peptide are changed [1,2]. By changing the corresponding codons upstream of the stop signal UGAA, we have analyzed if the -3 to -6 amino acids at the C-terminal region of the nascent peptide also affect termination. Lysine at position -3 gave increased readthrough, whereas a total of 28 variations at positions -4, -5, and -6 showed no significant effect on readthrough. The 3'-ends corresponding to the last six codons in 27 Escherichia coli genes were inserted upstream of a stop codon in the 3A' translation assay gene [1]. Readthrough of the stop codon was measured and a possible correlation with the Codon Adaptation Index (CAI) 131 of the respective genes was investigated. Sequences from genes with low CAI do not give any such correlation, whereas sequences from genes with high CAI values are correlated with high termination efficiency. This correlation disappears if the -1 and -2 codons/amino acids are changed. The results suggest that mainly the terminal dipeptide of the terminal hexapeptide sequence has an influence on termination in the tested E. coli genes. This influence is dependent on the charge of the -2 amino acid and is correlated with the alpha-helix propensity of the -1 amino acid, in accordance with results obtained from synthetic gene constructs [1,2].

Amino Acids↗

PubMed2004

Kinetic analysis of tRNA-directed transcription antitermination of the Bacillus subtilis glyQS gene in vitro.

Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNA(Gly) can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

5' Untranslated Regions↗

PubMed2002

Yeast polypeptide chain release factors eRF1 and eRF3 are involved in cytoskeleton organization and cell cycle regulation.

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. eRF1 recognizes nonsense codons UAA, UAG, and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRF1-dependent manner. In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively. Here we show that in yeast shortage of any one of the release factors was accompanied by a reduction in the levels of the other release factor and resulted in a substantial increase of nonsense codon readthrough. Besides, repression of the genes encoding these factors caused different effects on cell morphology. Repression of the SUP35 gene caused accumulation of cells of increased size with large buds. This was accompanied by the disappearance of actin cytoskeletal structures, impairment of the mitotic spindle structure, and defects in nuclei division and segregation in mitosis. The evolutionary conserved C-terminal domain of eRF3 similar to the elongation factor EF-1alpha was responsible for these effects. Repression of the SUP45 gene caused accumulation of unbudded cells with 2C and higher DNA content, indicating that DNA replication is uncoupled from budding. The data obtained suggest that eRF1 and eRF3 play additional, nontranslational roles in the yeast cell.

Actins↗

PubMed2002

Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli.

Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37 degrees C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.

Colon↗

PubMed2003

Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli.

The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.

Bacillus subtilis↗

PubMed2001

Regulation of immunity to the two-component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR.

Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream beta-galactosidase (beta-gal) fusion beyond an intragenic stem-loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the beta-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

Anti-Bacterial Agents↗

PubMed2001

The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon.

Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase. To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined. The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon. Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product. The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene. Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression. The amber codon position corresponded to a lysyl residue using either sequencing technique. The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes.

Amino Acid Sequence↗

PubMed1983

DNA restriction--modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription.

The EcoP1 and EcoP15 DNA restriction-modification systems are coded by the related P1 prophage and p15B plasmid. We have examined the organization of the genes for these systems using P1 itself, "P1-P15" hybrid phages expressing the EcoP15 restriction specificity of p15B and cloned restriction fragments derived from these phage DNAs. The results of transposon mutagenesis, restriction cleavage analysis. DNA heteroduplex analysis and in vitro transcription mapping allow the following conclusions to be drawn concerning the structural genes. (1) All of the genetic information necessary to specify either system is contained within a contiguous DNA segment of 5 x 10(3) bases which encodes two genes. One of them, necessary for both restriction and modification, we call mod and the other, required only for restriction (together with mod), we call res. (2) The res gene is about 2.8 x 10(3) bases long and at the heteroduplex level is largely identical for P1 and P15: it shows a small region of partial nonhomology and some restriction cleavage site differences. The mod gene is about 2.2 x 10(3) bases long and contains a 1.2 x 10(3) base long region of non-homology between P1 and P15 toward the N-terminus of the gene. The rest of the gene at this level of analysis is identical for the two systems. (3) Each of the genes is transcribed in vitro from its own promoter. It is possible that the res gene is also transcribed by readthrough from the mod promoter.

Coliphages↗

PubMed1992

cis-acting elements for in trans complementation of replication-defective mutant of tobacco mosaic virus.

We have shown that virus-encoded replicase components of tobacco mosaic virus (TMV)(130K/180K proteins) could complement a replication-defective mutant (LDR28) for viral replication in trans [Ogawa et al. Virology 185, 580-584 (1991)]. Using this trans-complementation system, we investigated the dispensability of regions of the 130K/180K protein genes as cis-acting elements for viral replication. A series of replication-defective mutants (LDRs) which had various deletions in the 130K/180K coding region were constructed. These were co-inoculated with a replication-competent mutant (LDCS29) into tobacco protoplasts. Accumulation of LDR-genomic RNA, CP mRNA, and CP was much increased by the removal of the 3' terminal one-third of the readthrough part of 180K protein gene (residues 4529-4937 of TMV-RNA) from LDR, suggesting that this region interferes with the viral replication in this trans-complementation system. In addition, accumulation of CP mRNA and CP was much decreased by the removal of the 5'terminal half of the 30K protein gene (residues 4938-5263 of TMV-RNA), suggesting the presence of an element to enhance the synthesis of CP mRNA in this region.

Blotting, Northern↗

PubMed1990

Transcriptional mapping of a DNA replication gene cluster in bacteriophage T4. Sites for initiation, termination, and mRNA processing.

A phage T4 genetic cluster that encodes DNA polymerase, several other DNA replication proteins, transcriptional factors, and the translation repressor RegA has been shown to be controlled by overlapping modes of transcription which initiate at several promoters. The promoters were mapped by using a combination of assays including Northern blotting, S1-mapping, RNA sequencing, and analysis of products of radioactive labeling of 5' ends on T4-induced RNA in vitro via the reaction catalyzed by eukaryotic guanylyl transferase (RNA capping assay). The most proximal in the cluster are two promoters that do not require any phage-induced factors for activation, i.e. they are T4 early promoters. Initiation at these promoters yields several RNA species having overlapping 5'-terminal sequences, the largest of which is estimated to be about 15,000 nucleotides long and to include all the cistrons of the cluster. A third early promoter maps inside the protein encoding segment of one of the cistrons (T4 gene 47), while at least five additional promoters map in intercistronic regions and are T4 middle promoters, i.e. they require the T4-induced DNA-binding transcription factor MotA. Transcriptional readthrough at a termination site within the T4 gene 45-44 intercistronic region is required for synthesis of gp44 and gp62, two essential T4 DNA-polymerase (gp43) accessory proteins. In contrast, transcription of T4 gene 43 is serviced by readthrough across a termination site in the regA-43 intercistronic region as well as by a MotA-dependent promoter that maps downstream of the termination site, and the region contains a site for processing by a T4-induced enzyme that also cleaves elsewhere in the polycistronic mRNA from the cluster (i.e. in the Shine-Dalgarno sequence of the gene 45.2 mRNA). The termination events in the gene 45-44 and regA-43 intercistronic regions both occur downstream of RNA stem-loop structures containing the sequence 5'CUUCGG3' in the loop segments. Transcription termination in the 78-base-pair regA-43 intercistronic region occurs about 60 nucleotides away from the gp43 initiator AUG, transcription initiation occurs at 38-40 nucleotides upstream from the AUG, and T4-dependent RNA processing occurs at several sites (including a GGAG sequence) between the transcription termination and initiation sites. Thus, all gp43-encoding mRNAs contain the translational operator (residues -40 to -1 relative to the AUG) for autogenous repression by this DNA polymerase (Andrake et al., 1988).(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1999

Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.

Bacterial Capsules↗

PubMed1982

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12.

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.

Bacterial Proteins↗