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PubMed1985

Antitermination is required for readthrough transcription of the maize rbcL gene by a bacteriophage promoter in Escherichia coli.

Sequences upstream of the 5' end of the rbcL gene in maize chloroplast fragment Bam 9, have a polar effect on the expression of rbcL from an external upstream bacteriophage PL promoter in Escherichia coli. This polarity can be suppressed by the bacteriophage transcription antitermination protein N or Q. The requirement for transcription antitermination is abolished if DNA upstream of rbcL is removed by a deletion. We have also investigated the ability of RNA polymerase initiating transcription at PL in the presence of N to transcribe through the normal rbcL transcription terminator and into sequences beyond. RNA polymerase initiating at PL can traverse rbcL and its 5' and 3'-flanking regions in the presence of N.

Base Sequence↗

PubMed1992

Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae.

The HSF1 gene of Saccharomyces cerevisiae directs the synthesis of the heat shock transcription factor, HSF. The gene is essential; disruption mutations are lethal. Using a plasmid shuffle screen, we isolated mutations in the HSF1 gene after in vitro mutagenesis of plasmid DNA with hydroxylamine. From a collection of both conditional (temperature-sensitive) and unconditional lethal mutations, we recovered mutations that map exclusively to the 5' half of the gene. All are nonsense mutations, including conditional mutations that map 5' to the portion of the HSF1 gene that encodes the DNA-binding domain of the transcription factor. For one such mutation, we demonstrated that the nonsense mutation is subject to translational readthrough, even though there are no known nonsense suppressors in the genetic background of our strain. Our results suggest that the HSF protein is highly tolerant of amino acid changes, a conclusion that is consistent with the very low degree of evolutionary conservation among HSF proteins. Our results also suggest that translational readthrough occurs with moderate efficiency in yeast, particularly when the terminator codon is followed immediately by an A or C residue. This result illustrates that the inference of gene function from mutant phenotype depends critically upon the analysis of a true null allele, and not merely an amber or ochre allele.

Amino Acid Sequence↗

PubMed1989

Genetic evidence for superoperonal organization of genes for photosynthetic pigments and pigment-binding proteins in Rhodobacter capsulatus.

Three adjacent operons, each concerned with photosynthesis in Rhodobacter capsulatus, have been shown by genetic means to be cotranscribable. In the course of describing the characteristics of the bchCA operon, which encodes two enzymes essential for bacteriochlorophyll synthesis, we found that the expression of the bchCA genes is influenced by readthrough from the upstream crtE and crtF genes. The crtE and crtF genes encode enzymes required for carotenoid biosynthesis and function as an operon. Furthermore, the distal structural gene of the bchCA operon, bchA, contains within it both the major oxygen-regulated promotor (Ppuf1) and the constitutive (Ppuf2) promotor for the puf operon. Since these three operons, crtEF, bchCA, and puf, are all transcribed in the same direction, it appears that polymerases traversing the downstream regions may start at any of several promoters. This pattern of transcription, which is unusual among bacteria, demonstrates that the activities of individual operons in a superoperonal cluster may be affected by their positions within the cluster.

Amino Acid Sequence↗

PubMed1992

Readthrough enhancement and promoter occlusion on the ribosomal genes of Xenopus laevis.

An RNA polymerase I termination site is found just upstream of the ribosomal gene promoter in mammals and amphibia. It has been suggested that this termination site may actively enhance ribosomal transcription in a process known as readthrough enhancement or that it may simply prevent the disruption of initiation complexes or promoter occlusion. There is, however, a consensus of opinion that the terminator is important for efficient ribosomal transcription. Here we have quantitatively investigated the relative importance of readthrough enhancement and promoter occlusion on the transcription of the microinjected Xenopus laevis ribosomal gene. The results show that, in this system, promoter occlusion is limited and terminator mutations predominantly affect readthrough enhancement. The terminator is shown to be unnecessary for the enhancer activity of the rest of the ribosomal spacer. Model calculations suggest that readthrough enhancement could be explained by polymerase recycling and that it may be unnecessary to postulate a specific mechanism of polymerase handover.

Animals↗

PubMed1990

In situ analysis of myelin basic protein gene expression in myelin-deficient oligodendrocytes: antisense hnRNA and readthrough transcription.

Mice homozygous for the recessive mutation myelin deficient (mld) exhibit a severe deficit in the synthesis of myelin basic protein (MBP). The primary defect in mld mice is an unusual rearrangement of the MBP gene. The mld MBP locus consists of two tandem MBP genes that span approximately 90 kb of DNA; the upstream gene contains an extensive inversion of its 3' region, while the downstream gene appears identical to the wild-type gene. In this report, the aberrant expression of the mld MBP locus was examined by in situ hybridization with MBP genomic and cDNA probes. In situ hybridization with a single-stranded genomic probe from the inverted region of the mld MBP gene revealed that primary transcripts initiating from the upstream MBP promoter elongate through the inverted region generating abundant antisense MBP heterogeneous nucleus RNA (hnRNA) transcripts in mld oligodendrocytes. Little or no antisense MBP RNA was detected in cytoplasmic regions in mld brain sections indicating that the antisense MBP hnRNA transcripts are not processed and transported out of the nucleus. Furthermore, we provide evidence that these abnormal transcripts elongate through the downstream MBP gene's promoter region, suggesting that transcription of the downstream mld MBP gene is inhibited by readthrough transcripts that originate at the upstream promoter.

Animals↗

PubMed1986

Transcription termination and processing sites in the bacteriophage lambda pL operon.

S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo. The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene. The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor. The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393). The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site). In the region between ral and Ea10, we mapped the 3' ends of three species of RNA. The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence. The terminator at this site may be partially N-resistant. In an RNase III deficient host, an additional RNA species is formed. The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence). In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence. These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA.

Bacteriophage lambda↗

PubMed1982

Overlap between ampC and frd operons on the Escherichia coli chromosome.

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.

Base Sequence↗

PubMed1992

The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the beta subunit of RNA polymerase.

The genes for the beta (rpoB) and beta' (rpoC) subunits of Escherichia coli RNA polymerase are the distal members of a complex transcriptional unit that contains four upstream ribosomal protein genes. The RNA polymerase subunit genes are transcribed at a lower frequency than the ribosomal protein genes as a result of termination at an attenuator preceding rpoB. A purified in vitro transcription system was developed using linear DNA templates that carry the attenuator. The ability of known termination and antitermination proteins to modulate termination at the attenuator was tested. Both NusA and NusG increase the frequency of transcriptional readthrough at the attenuator whereas NusB, S10, and Rho had no significant effect in this system.

Bacterial Proteins↗

PubMed2024

How do bacteria tune transcription termination efficiency?

Bacterial operons often contain intergenic transcription terminators that terminate some, but not all, RNA polymerase molecules. In these operons, the level of terminator readthrough determines downstream gene expression and helps establish protein ratios among co-regulated genes. Despite its critical role in maintaining stoichiometric gene expression, terminator strength remains difficult to predict from DNA sequence. The necessary features of a major class of bacterial terminators - intrinsic terminators - have been known for half a century, but a strong sequence-function model has yet to be developed. Here, we summarize high-throughput approaches for probing the sequence determinants of intrinsic termination efficiency and discuss the impact of trans-acting factors on this sequence-function relationship. Building on the main lessons from these studies, we map out the experimental challenges that must be circumvented to establish a quantitative model for termination efficiency.

Transcription Termination, Genetic↗

PubMed2000

Peptide nucleic acid-mediated PCR clamping as a useful supplement in the determination of microbial diversity.

Peptide nucleic acid (PNA)-mediated PCR clamping (H. Orum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, and C. Stanley, Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.

Bacteria↗

PubMed1984

Structural and functional characterization of Newcastle disease virus polycistronic RNA species.

Upon infection, the Newcastle disease virus (NDV) genome is transcribed to produce 18S, 22S, and 35S RNAs (M. Bratt , and W. Robinson, J. Mol. Biol. 23:1-21, 1967). The 22S RNA has been shown to contain 18S sequences and is thought to represent polycistronic transcripts generated by transcriptional readthrough of adjacent genes ( Varich et al., Acta Virol. 23:341-343, 1979). With improved extraction procedures, the 22S RNA was found to represent up to 25% of the total transcription in NDV-infected cells. This RNA was resolved into at least five discrete species on formaldehyde-agarose gels. All but one of these molecules contain 3' polyadenylate sequences but not internal polyadenylate sequences. These transcripts are found on polyribosomes of infected cells, suggesting that they are functional mRNAs.

Animals↗

PubMed2001

Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability.

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.

Amino Acid Sequence↗

PubMed1991

Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay.

A simple quantitative in vivo assay has been developed for measuring the efficiency of translation of one or other of the three termination codons. UAA, UAG and UGA in Saccharomyces cerevisiae. The assay employs a 3-phosphoglycerate kinase-beta-galactosidase gene fusion, carried on a multicopy plasmid, in which the otherwise retained reading frame is disrupted by one or other of the three termination codons. Termination readthrough is thus quantitated by measuring beta-galactosidase in transformed strains. Using these plasmids to quantitate the endogenous levels of termination readthrough we show that readthrough of all three codons can be detected in a non-suppressor (sup+) strain of S. cerevisiae. The efficiency of this endogenous readthrough is much higher in a [psi+] strain than in a [psi-] strain with the UGA codon being the leakiest in the nucleotide context used. The utility of the assay plasmids for studying genetic modifiers of nonsense suppressors is also shown by their use to demonstrate that the cytoplasmic genetic determinant [psi+] broadens the decoding properties of a serine-inserting UAA suppressor tRNA (SUQ5) to allow it to translate the other two termination codons in the order of efficiency UAA greater than UAG greater than UGA.

Antisense Elements (Genetics)↗

PubMed2001

Increased readthrough transcription across the simian virus 5 M-F gene junction leads to growth defects and a global inhibition of viral mRNA synthesis.

Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

Animals↗

PubMedDate not supplied

Expression vectors for quantitating in vivo translational ambiguity: their potential use to analyse frameshifting at the HIV gag-pol junction.

Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthrough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect. This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.

Amino Acid Sequence↗

PubMed1998

Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses.

The 3' terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3' non-coding region of 235 nucleotides, coat protein (CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages.

Amino Acid Sequence↗

PubMed1985

Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli.

Transcript mapping of the Escherichia coli sucAB, aceEF and lpd genes, encoding the five components of the pyruvate and 2-oxoglutarate dehydrogenase complexes, was carried out using single-stranded M13 probes. The sucA and aceE genes encode the specific dehydrogenase components (E1o, E1p), and the sucB and aceF genes encode the specific dihydrolipoamide acyltransferases (E2o, E2p). The common lipoamide dehydrogenase (E3) component is encoded by a single lpd gene adjacent to the aceEF genes. The sucAB, aceEF and lpd genes were all expressed on independent transcripts, and the promoters and terminators were identified. In addition, readthrough transcription from the sucAB genes to a downstream gene designated sucC, and from the aceEF genes to the adjacent lpd gene, was found. The relative levels of transcription of the suc, ace and lpd genes, and of the three different transcript types covering the ace-lpd region, were quantified using RNA from cells grown on different substrates. Most of the E3 components supplying the pyruvate dehydrogenase complex appear to be synthesised from approximately 6415-base aceEF-lpd readthrough transcripts, but additional approximately 4640-base aceEF transcripts terminating after the aceF gene provide a transcriptional basis for the observed stoichiometric excess of E1p and E2p relative to E3 in the assembled complex. Conversely most of the E3 components required for the 2-oxo-glutarate dehydrogenase complex appear to be synthesised from the independent 1670-base lpd transcripts.

Acyltransferases↗

PubMed2002

The protein synthesis inhibitors, oxazolidinones and chloramphenicol, cause extensive translational inaccuracy in vivo.

The oxazolidinone family is a new class of synthetic antibiotics that bind to the bacterial 50S ribosomal subunit. Two members of the family, linezolid and XA043, were examined for their effects on translational fidelity using a lacZ reporter gene in vivo. Both promoted highly significant frameshifting and nonsense suppression. Chloramphenicol, a peptidyl transferase inhibitor, affected translational fidelity in a similar fashion. Neither the oxazolidinones nor chloramphenicol stimulated misincorporation of amino acid residues at position 461 in the lacZ gene. In contrast, the aminoglycosides gentamicin and paromomycin, which interact with the decoding region of the 30S subunit, caused significant misincorporation but only modest increases in frameshifting or stop codon readthrough of the lacZ gene. We conclude that effects on translational fidelity may play a significant role in the mechanism of action of the oxazolidinones.

Acetamides↗