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PubMed1996

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

Animals↗

PubMed2006

Remodeling of the neuromuscular junction in mice with deleted exons 5 and 6 of acetylcholinesterase.

At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscular transmission (Minic et al., 2003). AChE knockout mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000). AChE and BChE genes encode two functional domains--the catalytic domain (exons 2, 3, and 4 of AChE, or exon 2 of BChE) and a C-terminal domain (exon 5 or 6 of AChE, or exon 3 of BChE)--that dictate the targeting of the enzymes (Massoulié, 2002). In mammals, the AChE gene produces three types of coding regions by deleting 5'- splice acceptor sites, which generate proteins; these proteins possess the same catalytic domain associated with distinct C-terminal peptides. AChE subunits of type R (readthrough) produce soluble monomers; they are expressed during development and are thought to be induced in the mouse brain by stress (Kaufer et al., 1998). AChE subunits of type H (hydrophobic) produce GPI-anchored dimers, mainly in blood cells. Subunits of type T (tailed) exist for both AChE and BChE. They represent the predominant AChE variant expressed in cholinergically innervated tissues (muscle and nerve). These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins ColQ (Krejci et al., 1997) and PRiMA (Perrier et al., 2002). At the NMJ, AChE is clustered by the interaction of the coding sequence of exon 6 with ColQ (Feng et al., 1999). The deletion of exons 5 and 6 in the AChE gene transforms anchored AChE into a soluble enzyme (Camp et al., 2004). The present study was designed to evaluate neuromuscular transmission and nicotinic ACh receptor (nAChR) distribution in muscles from mutant mice with deletions of these two spliced exons (AChE-del-exons-5+6-/-).

Acetylcholinesterase↗

PubMed1992

Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi.

The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).

Bacterial Proteins↗

PubMed1987

A human tRNA(iMet) gene produces multiple transcripts.

A third nonallelic locus of the human methionyl-tRNA multigene family (tRNA(iMet-3) was isolated. This gene, unlike two other tRNA(iMet) loci, lacks a remarkable run of T and C residues which functions as a termination of transcription signal. Instead, three tandem termination signals, each containing no more than four thymidylate residues, function as relatively inefficient termination signals. As a result, polymerase readthrough generates at least three transcripts in vitro. The efficiency of apparent termination varies significantly at these sites. All resulting transcripts appear to be processed in vitro.

Base Sequence↗

PubMed1987

Controlling mechanisms for expression of the bacteriophage T4 beta-glucosyltransferase gene.

The plasmid pTHF3047 carries a 2200 bp fragment containing the phage T4 beta-glucosyltransferase (beta gt) gene and part of the upstream gene 42 cloned in pBR313 under the control of the amp promoter P1. In T4-infected cells the beta gt gene may be expressed by a mechanism antagonizing Rho action. The plasmid pTHF3047 expressed about threefold higher beta-glucosyltransferase activity in a strain carrying the polarity-suppressing rho-102 mutation than in an otherwise isogenic rho+ strain, and production of T4 alpha gt- beta gt- phage was strongly stimulated. The plasmid copy number and the total T4-specific transcription was the same in the two strains. Two T4-specific transcripts from the plasmid, 600 bases and 1850 bases, were identified by Northern hybridization. Comparison with the T4 and plasmid maps suggested that both transcripts were initiated at P1, the 600 base transcript ending at the Rho-dependent terminator t42 between gene 42 and beta gt, and the 1850 base transcript reading through this terminator to the end of the beta gt gene. This analysis places t42 at position 25.1 on the T4 map, and a Rho-independent beta gt terminator at position 23.8. The three-fold higher beta gt expression in the rho- strain may be partially accounted for by Rho control of transcription. In the rho+ strain about half of the transcripts stopped at t42, while in the rho- strain readthrough appeared slightly higher. Thus, the t42 terminator was observed also on the plasmid, but appeared considerably less effective there than in the phage DNA in infected cells.

Enzyme Induction↗

PubMed1993

Characterization of the cyr1-2 UGA mutation in Saccharomyces cerevisiae.

cyr1-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyr1-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyr1-2 mutation. Construction of chimeric genes restricted the region containing the cyr1-2 mutation, and the cyr1-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyr1-2 mutation site result in null mutations. The complete open reading frame of the cyr1-2 gene expressed under the control of the GAL1 promoter complemented cyr1-d1 in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyr1-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.

Adenylyl Cyclases↗

PubMed1990

Escherichia coli cell division inhibitor DicF-RNA of the dicB operon. Evidence for its generation in vivo by transcription termination and by RNase III and RNase E-dependent processing.

We have established that the long non-coding intercistronic region of the dicB operon of Escherichia coli expresses a trans-acting division inhibitor specified by a region dicF, at most 65 nucleotides-long. The present study deals with the processing of dicBF operon mRNA in vivo, and identifies the dicF gene product as a 53 nucleotide RNA species. A sequence at the end of DicF resembles, and behaves as, a Rho-independent terminator, but further processing of readthrough transcripts, presumably by RNase III, followed by a limited 3' to 5' degradation, appears to generate additional DicF-RNA 3' ends. For the 5' end of DicF-RNA, our results show that a 190 nucleotide precursor DicF-RNA species is formed by cleavage at an RNase III site, while the 53 nucleotide minimal DicF-RNA is generated by further processing requiring the presence of an active form of RNase E in vivo. These data indicate that an untranslated product derived from an operon RNA can have a regulatory activity by affecting cell division.

Base Sequence↗

PubMed2003

Reverse transcriptase of Moloney murine leukemia virus binds to eukaryotic release factor 1 to modulate suppression of translational termination.

The pol (for polymerase) gene of the murine leukemia viruses (MuLVs) is expressed in the form of a large Gag-Pol precursor protein by the suppression of translational termination, or enhanced readthrough, of a UAG stop codon at the end of gag. A search for cellular proteins that interact with the reverse transcriptase of Moloney MuLV resulted in the identification of eRF1, the eukaryotic translation release factor 1. The proteins bound strongly in vitro, and the overexpression of eRF1 resulted in the RT-dependent incorporation of the protein into assembling virion particles. The overexpression of RT in trans enhanced the translational readthrough of a reporter construct containing the Gag-Pol boundary region. Noninteracting mutants of RT failed to synthesize adequate levels of Gag-Pol and could not replicate. These results suggest that RT enhances suppression of termination and that the interaction of RT with eRF1 is required for an appropriate level of translational readthrough.

Animals↗

PubMed2004

Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation.

Stress insults intensify fear memory; however, the mechanism(s) facilitating this physiological response is still unclear. Here, we report the molecular, neurophysiological and behavioral findings attributing much of this effect to alternative splicing of the acetylcholinesterase (AChE) gene in hippocampal neurons. As a case study, we explored immobilization-stressed mice with intensified fear memory and enhanced long-term potentiation (LTP), in which alternative splicing was found to induce overproduction of neuronal 'readthrough' AChE-R (AChE-R). Selective downregulation of AChE-R mRNA and protein by antisense oligonucleotides abolished the stress-associated increase in AChE-R, the elevation of contextual fear and LTP in the hippocampal CA1 region. Reciprocally, we intrahippocampally injected a synthetic peptide representing the C-terminal sequence unique to AChE-R. The injected peptide, which has been earlier found to exhibit no enzymatic activity, was incorporated into cortical, hippocampal and basal nuclei neurons by endocytosis and retrograde transport and enhanced contextual fear. Compatible with this hypothesis, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKCbetaII, accompanied by PKC-augmented LTP enhancement. Our findings demonstrate a primary role for stress-induced alternative splicing of the AChE gene to elevated contextual fear and synaptic plasticity, and attribute to the AChE-R splice variant a major role in this process.

Acetylcholinesterase↗

PubMed1987

Complete nucleotide sequence of a milk-transmitted mouse mammary tumor virus: two frameshift suppression events are required for translation of gag and pol.

We sequenced two recombinant DNA clones constituting a single provirus of the milk-transmitted mouse mammary tumor virus characteristic of BR6 mice. The complete provirus is 9,901 base pairs long, flanked by 6 base-pair duplications of cellular DNA at the site of integration. Five extensive blocks of open reading frame corresponding to the gag gene, the presumed protease, the pol and env genes, and the open reading frame orf within the long terminal repeat of the provirus were readily discernible. Translation of gag, protease, and pol involved three different translational reading frames to produce the three overlapping polyprotein precursors Pr77, Pr110, and Pr160 found in virus-infected cells. Synthesis of the reverse transcriptase and endonuclease therefore required two separate frameshifts to suppress the termination codons at the ends of the Pr77 and Pr110 domains. Direct evidence is presented for translational readthrough of both stop codons in an in vitro protein synthesis system.

Amino Acid Sequence↗

PubMed1989

Transcription elongation factor SII (TFIIS) enables RNA polymerase II to elongate through a block to transcription in a human gene in vitro.

Elongation and termination by RNA polymerase II are important regulatory steps for eukaryotic gene expression. We have previously studied the transcription of linear DNA templates where specific initiation of transcription by highly purified RNA polymerase II can be achieved in the absence of promoters and promoter-specific factors. Using these templates we have shown that a human histone gene, H3.3, contains sequences (intrinsic terminators) within which purified RNA polymerase II will efficiently terminate transcription (Reines, D., Wells, D., Chamberlin, M.J., and Kane, C. M. (1987) J. Mol. Biol. 196, 299-312). Curiously, these signals were found within an intron, 3'-untranslated, and protein-encoding regions of the gene suggesting that they might act to attenuate transcription of H3.3 in vivo. Here we show that intrinsic terminator sequences from an H3.3 gene intron also block in vitro transcript elongation by RNA polymerase II when the enzyme has initiated transcription from a promoter using highly purified transcription initiation factors. However, under the conditions used for promoter-specific transcription there is little transcript release. Instead the polymerase can pause at these sites for periods exceeding 60 min. We have identified and partially purified an activity from HeLa cells that causes the transcription complex to read through this block to transcription elongation. This readthrough activity fractionates with a previously characterized elongation factor (SII) over three chromatographic columns. A homogeneous preparation of calf thymus SII can also provide this activity in trans. This factor may facilitate passage of the RNA polymerase II transcription complex through such intragenic sites in cellular genes in vivo.

Animals↗

PubMed2004

CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression.

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

Bacillus subtilis↗

PubMed1994

Paramyxovirus mRNA editing leads to G deletions as well as insertions.

Paramyxoviruses are thought to edit their P gene mRNAs co-transcriptionally, by a mechanism in which the polymerase stutters and reads the same template base more than once. Sendai virus (SeV) and bovine parainfluenza virus type 3 (bPIV3) are closely related viruses, but SeV edits its P gene mRNA with the insertion of a single G residue (at approximately 50% frequency) within the sequence 5' A6G3, whereas bPIV3 inserts 1 to approximately 6 Gs at roughly equal frequency within the sequence 5' A6G4. When SeV synthetic mini-genomes containing either SeV or bPIV3 P gene editing cassettes are expressed from cDNA in cells which are also transfected with the SeV NP, P and L genes, the virus-specific editing patterns were reproduced. Since the bPIV3 editing pattern was reproduced in a system that is otherwise completely SeV, this suggests that all the information for the virus-specific editing patterns is due to the RNA sequence itself. Unexpectedly, the length of the template C run was found to be critical, even though it varies from 3 to 7 nucleotides in length in different viruses. Expanding this template C run first led to attenuation of the insertion phenotype, and then to deletions rather than insertions. A stuttering or slippage model to account for these events has been further refined to include a pressure which displaces the nascent strand in a given direction once it has disengaged from the template, and the similarities of this model to those which account for readthrough of cellular RNA polymerase transcription blocks are discussed.

Base Sequence↗

PubMed1995

Transcription termination of the streptokinase gene of Streptococcus equisimilis H46A: bidirectionality and efficiency in homologous and heterologous hosts.

In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes.

Base Sequence↗

PubMed1997

In vivo and in vitro evidence for an anti-Rho activity induced by the phage P4 polarity suppressor protein Psu.

The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity in Escherichia coli by overcoming Rho termination factor activity. Two new psu mutants defective in polarity suppression are described. The psu5 mutation deletes codons 95-98 from about the middle of the gene, and the mutant protein is inactive. The psu6 mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression of psu+ from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at the rho attenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defective rho mutants. The polarity suppression activity induced by Psu is demonstrated in vitro by the efficient readthrough of Rho-dependent terminators lambda tR1 and TIS2 during coupled transcription-translation. Purified Rho protein restores termination at TIS2 when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.

Capsid↗

PubMed1992

A possible role for nonsense suppression in the synthesis of a human cytomegalovirus 58-kDa virion protein.

A 1.6-kb late mRNA originating from the HindIII R fragment of human cytomegalovirus (HCMV) encodes a 58-kDa virion phosphoprotein. Data presented support the hypothesis that this protein may be synthesized via the translational readthrough of an opal termination codon separating two open reading frames located in tandem. To our knowledge this is the first report of nonsense suppression as a means of regulating gene expression in HCMV.

Amino Acid Sequence↗

PubMed1999

Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription.

M2-1 protein of human respiratory syncytial virus (RSV) is a transcription antitermination factor that is important for the efficient synthesis of full-length mRNAs as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of nonsegmented negative-strand RNA viruses. The contributions of these effects to RSV sequential transcription were investigated with minigenomes which contained one to five genes which were either foreign marker genes or authentic RSV genes. When evaluated on a promoter-proximal gene, the effect of M2-1 on the synthesis of full-length mRNA was much greater for a long (1,212- or 1,780-nucleotide) gene (up to a 615-fold increase) than for a short (274-nucleotide) gene (less than a 2-fold increase). This was independent of whether the gene contained non-RSV or RSV-specific sequence. Once the polymerase had terminated prematurely, it was unable to reinitiate at a downstream gene. These studies also confirmed that M2-1 enhances the synthesis of polycistronic mRNAs and that the magnitude of this effect varied greatly among different naturally occurring gene junctions. The synthesis of polycistronic mRNAs, which presumably involves antitermination at the gene-end signal, required a higher level of M2-1 than did the synthesis of the corresponding monocistronic mRNAs. M2-1 did not have a comparable antitermination effect at the junction between the leader region and the first gene. In a minigenome containing the NS1 and NS2 genes in their authentic sequence context, synthesis of full-length NS1 and NS2 mRNAs in the absence of M2-1 was remarkably high (36 and 57%, respectively, of the maximum levels observed in the presence of M2-1). In contrast, synthesis of mRNA from additional downstream genes was highly dependent on M2-1. Thus, RSV has the potential for two transcription programs: one in the absence of M2-1, in which only the NS1 and NS2 genes are transcribed, and one in the presence of M2-1, in which sequential transcription of the complete genome occurs. The dependence on M2-1 for transcription was greater for a gene in the fifth position from the promoter than for one in the third position. This indicates that under conditions where M2-1 is limiting, its concentration affects the gradient of transcription. Although M2-1 was found to have profound effects on transcription, it had no effect on replication of any minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication. Finally, since a permissive RSV infection is marked by a gradual increase in the intracellular accumulation of viral proteins including M2-1, we examined the relative abundances of various mRNAs during RSV infection for evidence of temporal regulation of transcription. None was found, implying that the availability of M2-1 during a permissive infection is sufficient at all times such that its concentration does not mediate temporal regulation of gene transcription.

Gene Expression Regulation, Viral↗

PubMed1988

Ribosome release modulates basal level expression of the trp operon of Escherichia coli.

The leader peptide stop codon (UGA) of the Escherichia coli trp operon was replaced by UAA and UAG. The transcriptional behavior of the mutated leader regions in vitro and the extent of transcription termination observed with each in vivo were virtually identical to that of the wild type leader region. Introduction of a release factor 1 (UAA- and UAG-specific) mutation into strains with the different stop codons caused increased termination in strains with UAA and UAG, but not with UGA (in cells grown in the presence of tryptophan). This finding provides evidence for the view that ribosome release from the leader peptide stop codon is an important event in setting the basal level of transcription readthrough at the trp attenuator.

Amino Acid Sequence↗