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PubMed1986

Characterization of messenger RNAs of measles virus.

Ten different RNA species associated with measles virus were detected in virus-infected cells. The largest RNA (no. 1) was considered to be genomic RNA and the other nine RNAs were found to have a methylated cap as well as a poly(A) tail. Northern blot hybridization indicated that RNAs no. 2 and 7 to 10 correspond to mRNAs encoding structural proteins, and that RNAs no. 3 to 6 are intermediate-sized (IS)RNAs. The possibility that the ISRNAs represent defective interfering RNA or a precursor form of the mRNA seems to be unlikely since the amounts of IS RNAs did not increase after undiluted passages or decrease in a pulse-chase experiment. These results indicate that IS RNAs are readthrough transcripts of neighbouring cistrons.

Genes, Viral↗

PubMed2005

Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons.

Incorporation of the 21st amino acid, selenocysteine, into proteins is specified in all three domains of life by dynamic translational redefinition of UGA codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3'UTR of selenoprotein mRNAs. Here we present comparative sequence analysis and experimental data supporting the presence of a second stop codon redefinition element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal gene, SEPN1. This element is sufficient to stimulate high-level (6%) translational redefinition of the SEPN1 UGA codon in human cells. Readthrough levels further increased to 12% when tested in the presence of the SEPN1 3'UTR SECIS. Directed mutagenesis and phylogeny of the sequence context strongly supports the importance of a stem loop starting six nucleotides 3' of the UGA codon. Sequences capable of forming strong RNA structures were also identified 3' adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH, SelO, and SelT selenoprotein genes.

Animals↗

PubMed1987

Complete nucleotide sequence of the matrix protein mRNA and three intergenic junctions of human parainfluenza virus type 3.

The complete sequence of the gene encoding the matrix protein (M) of human parainfluenza virus type 3 (PIV-3) was determined from cDNA clones and from primer extension dideoxy sequencing of the viral genome. The M mRNA is 1150 nucleotides in length, exclusive of polyadenylate, and codes for a protein of 353 amino acids, having a calculated molecular weight of 39,480. The M protein of PIV-3 was found to have a high degree of sequence homology with that of a closely related paramyxovirus, Sendai virus, and to a lesser extent it contained sequence homology with two more distant paramyxoviruses, measles virus and canine distemper virus. We also determined the sequences of the intergenic junctions for the first four genes of PIV-3: NP, P, M, and F. Comparison of these sequences yielded a consensus mRNA start sequence of 5'-AGGANNAAAGA-3', an mRNA end sequence of 5'-UAAGAAAAA-3', and an intergenic sequence of 5'-CUU-3'. The end sequence of the M gene is unusual in that it contains an eight base insertion prior to the A5 tract found in the consensus sequence. This disruption appears to cause a high frequency of readthrough by the viral transcriptase at this junction.

Amino Acid Sequence↗

PubMed1996

Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2).

An internal segment of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. relA lies downstream of a gene (apt) that apparently encodes adenine phosphoribosyltransferase and is transcribed from two promoters, relAp1 and relAp2, and by transcriptional readthrough from apt. While the level of relAp2 transcripts remained relatively constant, relAp1 activity apparently peaked during transition phase, following a decline in readthrough transcription from apt. Disruption of relA using an att- derivative of the temperate phage phi C31 abolished ppGpp synthesis on amino acid depletion. When grown on agar, the disruptants grew more slowly than a control lysogen made with an att+ phage vector and gave smaller colonies that sporulated normally. The relA mutation had no consistent or marked effect on actinorhodin production in either liquid- or agar-grown cultures, indicating that elevated levels of (p)ppGpp are not essential for triggering the onset of antibiotic production.

Amino Acid Sequence↗

PubMed1995

Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

Animals↗

PubMed2006

Translational control in positive strand RNA plant viruses.

The great variety of genome organizations means that most plant positive strand viral RNAs differ from the standard 5'-cap/3'-poly(A) structure of eukaryotic mRNAs. The cap and poly(A) tail recruit initiation factors that support the formation of a closed loop mRNA conformation, the state in which translation initiation is most efficient. We review the diverse array of cis-acting sequences present in viral mRNAs that compensate for the absence of a cap, poly(A) tail, or both. We also discuss the cis-acting sequences that control translation strategies that both amplify the coding potential of a genome and regulate the accumulations of viral gene products. Such strategies include leaky scanning initiation of translation of overlapping open reading frames, stop codon readthrough, and ribosomal frameshifting. Finally, future directions for research on the translation of plant positive strand viruses are discussed.

3' Untranslated Regions↗

PubMed1982

Nucleotide sequence of tobacco mosaic virus RNA.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.

Amino Acid Sequence↗

PubMed1996

The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region.

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta-globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3'UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3'UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3'UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3'UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3'UTR, whereas translation into the alpha-globin mRNA 3'UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3'UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3'UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha-globin mRNA 3'UTR stability element.

Animals↗

PubMed1982

Effect of rifampicin on expression of lacZ fused to promoters or terminators of the E.coli rpoBC operon.

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli lie downstream of at least two ribosomal protein genes in a single unit of transcription. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can strongly stimulate transcription of the polymerase genes, while activating the neighbouring ribosomal genes only slightly. We have investigated the mechanism of this effect by fusing strong promoters, a weak internal promoter, and an attenuator of the polymerase operon to the lacZ gene, in derivatives of plasmid pMC81. Studies of these fusions confirm our conclusion, based on similar fusions to galK, that rifampicin can foster readthrough of transcriptional terminators. They also suggest the existence of extra terminators and anti-termination elements in the above transcription unit.

DNA Restriction Enzymes↗

PubMed1995

Sequence analysis and gene content of potato mop-top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses.

The complete sequence of the 2315 nucleotides in RNA 3 of potato mop-top furovirus (PMTV) isolate T was obtained by analysis of cDNA clones and by direct RNA sequencing. The sequence contains an open reading frame for the coat protein (20K) terminated by an amber codon, followed by an in-phase coding region for an additional 47K. PMTV therefore resembles soil-borne wheat mosaic (SBWMV) and beet necrotic yellow vein (BNYVV) viruses (two other fungus-transmitted viruses with rod-shaped particles) in having a coat protein-readthrough product. Comparison of the 3' untranslated regions of PMTV RNA 2 and RNA 3 reveals a long conserved block of 150 nucleotides, which contains two repeated sequences and has the potential to form consecutive pseudoknot structures. PMTV RNA 3 ends a few nucleotides downstream of this conserved block, but RNA extends for a further 140 nucleotides, which can potentially form a 3'-terminal tRNA-like structure similar to those in the RNA species of SBWMV, tymoviruses, and some tobamoviruses. PMTV coat protein has amino acid sequence identities of 30 and 28% with SBWMV and BNYVV coat proteins, respectively, and apparent structural similarities with tobacco mosaic virus coat protein. The coat protein readthrough domains of PMTV, SBWMV, and BNYVV have shared residues throughout their length but no extended sequences are conserved. The presence in RNA 3 of coding sequences for only the coat protein and readthrough domain distinguishes PMTV from SBWMV and BNYVV, both of which have them in RNA 2 along with one or more other genes. Comparison of the genomes of PMTV, BNYVV, and SBWMV shows that furoviruses exhibit considerable heterogeneity in genome organization.

Amino Acid Sequence↗

PubMed2004

An internal rearrangement in an Arabidopsis inverted repeat locus impairs DNA methylation triggered by the locus.

In plants, transcribed inverted repeats trigger RNA interference (RNAi) and DNA methylation of identical sequences. RNAi is caused by processing of the double-stranded RNA (dsRNA) transcript into small RNAs that promote degradation of complementary RNA sequences. However, the signals for DNA methylation remain to be fully elucidated. The Arabidopsis tryptophan biosynthetic PAI genes provide an endogenous inverted repeat that triggers DNA methylation of PAI-identical sequences. In the Wassilewskija strain, two PAI genes are arranged as a tail-to-tail inverted repeat and transcribed from an unmethylated upstream promoter. This locus directs its own methylation, as well as methylation of two unlinked singlet PAI genes. Previously, we showed that the locus is likely to make an RNA signal for methylation because suppressed transcription of the inverted repeat leads to reduced PAI methylation. Here we characterize a central rearrangement in the inverted repeat that also confers reduced PAI methylation. The rearrangement creates a premature polyadenylation signal and suppresses readthrough transcription into palindromic PAI sequences. Thus, a likely explanation for the methylation defect of the mutant locus is a failure to produce readthrough dsRNA methylation triggers.

Arabidopsis↗

PubMed1990

Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons.

Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine. Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli. To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator. This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E. coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization. This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis. A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.

Animals↗

PubMed1990

tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.

Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.

Chromosome Deletion↗

PubMed1998

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3,803 nucleotides and has five open reading frames (ORFs). The 5' ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

Amino Acid Sequence↗

PubMed2000

A member of the Plasmodium falciparum Pf60 multigene family codes for a nuclear protein expressed by readthrough of an internal stop codon.

Four large multigene families have been described in Plasmodium falciparum malaria parasites (var, rif, stevor and Pf60). var and rif genes code for erythrocyte surface proteins and undergo clonal antigenic variation. We report here the characterization of the first Pf60 gene. The 6.1 gene is constitutively expressed by all mature blood stages and codes for a protein located within the nucleus. It has a single copy, 7-exon, 5' domain, separated by an internal stop codon from a 3' domain that presents a high homology with var exon II. Double-site immunoassay and P. falciparum transient transfection using the reporter luciferase gene demonstrated translation through the internal ochre codon. The 6.1 N-terminal domain has no homology with any protein described to date. Sequence analysis identified a leucine zipper and a putative nuclear localization signal and showed a high probability for coiled coils. Evidence for N-terminal coiled coil-mediated protein interactions was obtained. This identifies the 6.1 protein as a novel nuclear protein. These data show that the Pf60 and var genes form a superfamily with a common 3' domain, possibly involved in regulating homo- or heteromeric interactions.

Amino Acid Sequence↗

PubMed2000

Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10.

Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides+259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone-responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Qa promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion analyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction.

Amino Acid Sequence↗

PubMed1995

Genes and cis-acting sequences involved in replication of barley yellow dwarf virus-PAV RNA.

Deletion and point mutation analyses were employed to determine gene products and cis-acting signals involved in translation, replication, and encapsidation of barley yellow dwarf virus (PAV serotype) RNA in oat protoplasts. Of the six open reading frames (ORFs), only ORFs 1 and 2, which include the putative RNA-dependent RNA polymerase gene, were required for replication. In vitro translation of these mutants revealed that sequence upstream of the shifty heptanucleotide was required for ribosomal frameshifting, and that a 3'-translational enhancer stimulated translation more efficiently when located in closer proximity to the translated ORFs. Deletion of the coat protein gene reduced the accumulation of genomic but not subgenomic RNA. The carboxy-terminally extended form of the coat protein, produced by readthrough of its stop codon, was not required for encapsidation. Although the ORF6 product was not necessary, cis-acting RNA signals in and around ORF6 were required for RNA replication. Defective RNAs harboring various deletions were not replicated in trans by the co-inoculated wild-type helper genome, suggesting that replication of PAV RNA may be coupled to translation.

Avena↗

PubMed1991

Identification and characterization of a C-terminally extended form of recombinant murine IL-6.

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.

Amino Acid Sequence↗