Single Transcriptional Unit Research Articles

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel.

The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide- and dipeptide-binding protein-dependent transport systems from several Gram-negative and Gram-positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP-binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel-containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding-protein-dependent transport system for nickel.

The methyl-accepting transducer protein HtrI is functionally associated with the photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium.

We have investigated the functional relationship between two proteins involved in the photosensory system of the archaeon Halobacterium salinarium: the photoreceptor sensory rhodopsin I (SRI) and the halobacterial transducer rhodopsin I (HtrI), which has been proposed to be the putative signal transducer of SRI, by genomic DNA analysis of two independent SRI negative mutants, Pho81 and D1. Southern and PCR analyses revealed that both strains bear alterations in the 5' flanking region of the gene encoding SRI, sopI. DNA sequence analysis confirmed the occurrence in this region of htrI, the gene encoding the putative transducer protein. PCR and Northern analyses have shown further that sopI and htrI are expressed as a single transcriptional unit, thus explaining the lack of SRI in mutants with a defective htrI. Expression of the cloned sopI under the control of a heterologous promoter did not restore the SRI-dependent photoresponse in the strain Pho81. Moreover, the photocycling rate of the expressed pigment was clearly lower than in wild type. HtrI is therefore essential for SRI function and most likely modulates the photochemical properties of the photoreceptor via direct physical interaction. Finally, reintroduction of both sopI and htrI into Pho81 and D1 restored the SRI photochemistry and its physiological function. Our results provide the first experimental evidence for the functional coupling between SRI and HtrI and corroborate the proposed model in which HtrI acts as the signal transducer of this archaeal seven-helix photoreceptor in a way analogous to the bacterial chemotaxis transducers.

Insertional mutagenesis, cloning and expression of gentisate pathway genes from Pseudomonas alcaligenes NCIB 9867

An artificial transposon, Omegon‐km, was used for insertional mutagenesis of Pseudomonas alcaligenes NCIB 9867. Insertion mutants were recovered at approximately 6 times 10‐6 per recipient cell; 12.6% of the Omegon‐km insertion mutants were catabolic mutants deficient in gentisate pathway enzymes. A specific insertion mutant, p44S, was found to lack two gentisate pathway enzymes, viz. xylenol methylhydroxylase and 3‐hydroxy‐4‐methylbenzoate specific 6‐hydroxylase, but expressed low levels of gentisate 1,2‐dioxygenase. Chromosomal DNA encoding gentisate pathway genes adjacent to Omegon‐km insertions were subcloned into a pRK415 vector and expressed in a cured derivative of Ps. putida NCIB 9869. Removal of both transcriptional and translational stop signals by subcloning was found to relieve suppression of all three enzymes, thus confirming the polar nature of the insertion. It was postulated that both xylenol methylhydroxylase and 6‐hydroxylase were present together in a single transcriptional and translational unit, separate from gentisate 1,2‐dioxygenase. The use of Omegon‐km, a transposable element for in vivo insertional mutagenesis, has enabled the rapid cloning of gentisate pathway genes and the construction of a restriction map comprising of a gene cluster involving at least three enzymes for 2,5‐xylenol degradation.

In vivo formation of active aspartate transcarbamoylase from complementing fragments of the catalytic polypeptide chains.

Despite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains. Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed. When the N-terminal peptide (residues 1-242) and the C-terminal peptide (residues 235-310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts. Mixing the two insoluble pellets in 6.5 M urea, followed by a 10-fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8-amino acid redundancy. When the two partial genes were linked into a single transcriptional unit separated by a 15-nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains. The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (Vmax) only slightly less than that of the wild-type C trimer and ATCase. However, Km for aspartate exhibited by the C trimer composed of fragmented chains is more than 10-fold larger than that of the wild-type trimer. The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild-type ATCase for which the Hill coefficient is 1.7. Km for aspartate as well as Kd for the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate are significantly higher than the analogous values for wild-type ATCase. Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild-type ATCase.

Two Adenovirus Proteins with Redundant Activities in Virus Growth Facilitates Tripartite Leader mRNA Accumulation

Most adenovirus-specific mRNAs expressed late after infection originate from a single transcription unit, the socalled major late transcription unit. All mRNAs expressed from this unit have in common a 201 nucleotide-long spliced tripartite leader segment attached to their 5′ ends. Human adenovirus mutants that carry large deletions in early region 4 (E4) are severely defective in expression of nuclear and cytoplasmic RNA derived from the major late transcription unit. We have previously shown that E4 post-transcriptionally stimulates accumulation of tripartite leader containing mRNAs by a mechanism that requires an intron in the nuclear precursor RNA. To identify the E4 products responsible for this stimulatory effect on tripartite leader mRNA accumulation, we constructed CMV expression vectors encoding single E4 open reading frames (ORFs). By comparing the activity of these plasmids in a transient DNA cotransfection assay we could show that both the E4-ORF3 and E4-ORF6 proteins individually are able to stimulate mRNA accumulation from tripartite leader intron containing transcription units. Furthermore, this stimulatory activity was not dependent on coexpresaion of other viral gene products. These results are interesting since the same two E4 proteins have been shown to have interchangeable activities in lytic infection, and expression of one has been suggested to be sufficient to substitute for the whole E4 region for virus growth. Finally, we show that the absence of E4 expression during a virus infection results in abnormalities in tripartite leader assembly. This result suggests that E4 proteins may be required for efficient tripartite splicing also during a lytic virus infection.

Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli

The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea-associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papI genes of the P fimbrial adhesin, respectively. The 5' termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine-responsive regulatory protein (Lrp)-binding sites which contained differentially methylated GATC sequences, a cAMP-CRP-binding site, and an integration host factor (IHF)-binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.

Human biliary glycoprotein gene: characterization of a family of novel alternatively spliced RNAs and their expressed proteins.

Eight different human biliary glycoprotein (BGP) isoantigens, structurally related members of the carcinoembryonic antigen family, CD66/67 family, and immunoglobulin superfamily, are derived by alternative splicing from a single genomic transcription unit. Novel BGP isoforms have been identified by polymerase chain reaction amplification and by DNA sequencing of amplified cDNA segments. In addition to verifying previously documented BGPs, we describe four new forms, two of which have unusual nonimmunoglobulin exons contributed by inverted Alu repeats. Determination of the genomic DNA sequence encompassing most of the known extracellular and intracellular domains demonstrates that the translatable Alu-like sequences are encoded in bona fide exons. The third novel BGP isoform contains none of the extracellular disulfide-linked immunoglobulin-like domains typical of these molecules but retains N-terminal and intracellular domains, suggesting distinct functions for N-terminal versus other disulfide-linked domains. cDNAs coding for each identified isoform have been transfected into COS7 monkey cells, and the resulting polypeptides are heavily N glycosylated but can be deglycosylated to their expected primary sizes. Many of these deglycosylated forms can be correlated with unique patterns of BGP expression in different cell lines, while in granulocytes, some previously undescribed or alternatively modified forms may predominate. The BGP family represents a potentially large but unknown source of functional diversity among cells of epithelial and hematopoietic origin. The availability of a defined set of expressed of BGP cDNAs should permit critical definition of their function.

Human biliary glycoprotein gene: characterization of a family of novel alternatively spliced RNAs and their expressed proteins.

Eight different human biliary glycoprotein (BGP) isoantigens, structurally related members of the carcinoembryonic antigen family, CD66/67 family, and immunoglobulin superfamily, are derived by alternative splicing from a single genomic transcription unit. Novel BGP isoforms have been identified by polymerase chain reaction amplification and by DNA sequencing of amplified cDNA segments. In addition to verifying previously documented BGPs, we describe four new forms, two of which have unusual nonimmunoglobulin exons contributed by inverted Alu repeats. Determination of the genomic DNA sequence encompassing most of the known extracellular and intracellular domains demonstrates that the translatable Alu-like sequences are encoded in bona fide exons. The third novel BGP isoform contains none of the extracellular disulfide-linked immunoglobulin-like domains typical of these molecules but retains N-terminal and intracellular domains, suggesting distinct functions for N-terminal versus other disulfide-linked domains. cDNAs coding for each identified isoform have been transfected into COS7 monkey cells, and the resulting polypeptides are heavily N glycosylated but can be deglycosylated to their expected primary sizes. Many of these deglycosylated forms can be correlated with unique patterns of BGP expression in different cell lines, while in granulocytes, some previously undescribed or alternatively modified forms may predominate. The BGP family represents a potentially large but unknown source of functional diversity among cells of epithelial and hematopoietic origin. The availability of a defined set of expressed of BGP cDNAs should permit critical definition of their function.

SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

Nucleotide sequence of the nfa A gene encoding the antigen 8786 adhesive factor of enterotoxigenicEscherichia coli

The nucleotide sequence of a 714-bp DNA fragment containing the enterotoxic Escherichia coli (ETEC) adhesive factor 8786 structural gene, designated nfaA, revealed an open reading frame of 498 bp encoding a polypeptide of 166 amino acids. Primer-extension experiments showed that the nfaA gene is within a single transcription unit. No homology was found with the ETEC adhesive factors already sequenced. In contrast, a homology with Salmonella enteritidis fimbrin SEF 14 was observed.

Nucleotide sequence of the nfa A gene encoding the antigen 8786 adhesive factor of enterotoxigenic Escherichia coli

The nucleotide sequence of a 714-bp DNA fragment containing the enterotoxic Escherichia coli (ETEC) adhesive factor 8786 structural gene, designated nfaA, revealed an open reading frame of 498 bp encoding a polypeptide of 166 amino acids. Primer-extension experiments showed that the nfaA gene is within a single transcription unit. No homology was found with the ETEC adhesive factors already sequenced. In contrast, a homology with Salmonella enteritidis fimbrin SEF 14 was observed.

Expression of the Chlamydomonas reinhardtii chloroplast tRNA Glu gene in a homologous in vitro transcription system is independent of upstream promoter elements

Chloroplast tRNA Glu is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA Glu cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 m m KCl, 9 m m MgCl 2 and with 25 μg plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of α-amanitin (500 μg/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA Glu gene copies found in the C. reinhardtii chloroplast genome ( trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5′ region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the “−10 and −35 region” and the “ Euglena promoter” types. Surprisingly, deletion of the complete 5′ region did not affect in vitro transcription, while partial deletions of the 5′ and 3′ coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA Glu gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.

Biosynthesis of the erythromycin macrolactone and a rational approach for producing hybrid macrolides

The three eryA genes involved in the formation of the polyketide portion of the macrolide antibiotic erythromycin in Saccharopolyspora erythraea, appear to be organized in a single transcriptional unit on the basis of the results of gene disruption experiments. An insertion sequence-like element of lower G + C content separates eryAI from eryAII. The organization of the enzymatic domains present in the eryA-encoded multifunctional polypeptides, determined by computer-assisted analysis, is presented. This has enabled the determination of a putative dehydratase domain. A rational approach for producing novel macrolides by introducing selected changes in polyketide synthase genes is outlined. The isolation of a lactone intermediate resulting from an early synthesis step in macrolactone formation is also presented.

Structural analysis of ternary complexes of Escherichia coli RNA polymerase : Deoxyribonuclease I footprinting of defined complexes

The structure and properties of ternary complexes of RNA polymerase are of central importance in understanding the mechanisms of transcriptional elongation and termination, and the regulation of these primary steps in gene expression. However, there has been no systematic study of the structure and properties of such complexes along a single transcription unit. Recently, we have described the isolation of a collection of halted ternary complexes of Escherichia coli RNA polymerase bearing transcripts from 11 to 35 nucleotides in length along two different transcription units (accompanying paper). Here, we report structural studies of these complexes using DNase I footprinting. Surprisingly, nearly all of the different ternary complexes have distinctly different footprints along the two DNA strands, and the position of the footprint relative to the 3′ end of the transcript also varies for most complexes. Halted complexes bearing transcripts of comparable size do not have identical footprints; hence, DNA sequence as well as transcript length plays a role in determining the size and position of the footprint. These differences in structure are consistent with our earlier findings that ternary complexes can differ considerably in stability and gel mobility. The downstream boundary of the RNA polymerase in ternary complexes does not move forward regularly as successive nucleotide residues are added to the RNA chain. In contrast, the upstream boundary moves forward more or less in concert with the movement of the 3′ terminus of the transcript. These factors lead to a general compression of the overall footprint as transcription proceeds, together with a steady movement of the 3′ terminus of the RNA toward the downstream boundary of the polymerase. Ultimately, after the length of the RNA transcript has increased from eight to ten nucleotides, the downstream boundary of the complex is found to move downstream along the DNA, suggesting a translocation event. We suggest that RNA chain elongation, like RNA chain initiation, may involve a saltatory process in which net translocation of the complex along the DNA occurs only after addition of a number of ribonucleotides to the RNA chain.

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle.

Protein kinase C as a mediator of TSH and thyroid autoantibody action.

Although it is well-established that TSH activates a cAMP-dependent pathway in the thyroid follicular cell leading to thyroid hormone synthesis and release, the present review provides new evidence that TSH also activates a non-cAMP-dependent signal transduction system. This cascade involves phosphoinositide (PI) turnover, diacylglycerol accumulation and protein kinase C (PKC) activation. Activation of this pathway leads to an inhibition of differentiated thyroid function in vitro. Recent evidence suggests that TSH can activate both pathways via a single transcription unit. Unlike TSH, TSH-receptor antibodies may selectively activate cAMP with no effects on PI turnover. In contrast, preliminary studies suggest TSH-blocking antibodies may activate PKC. PKC may be an important mediator of TSH and, possibly, thyroid autoantibody action.

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnfENX. The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+). However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated. AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions.

The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase

The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin-resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzylviologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.

PEXPRESS: A family of expression vectors containing a single transcription unit active in prokaryotes, eukaryotes and in vitro

We have constructed a family of expression vectors containing a single transcription unit that is active in Escherichia coli, eukaryotic cells, and in coupled in vitro transcription-translation systems. These vectors use the Rous sarcoma virus-long terminal repeat (RSV-LTR) as the promoter/enhancer for eukaryotic cells. In vitro transcription is made possible by inclusion of a bacteriophage T7 promoter. This same promoter is actively transcribed in E. coli that produce T7 RNA polymerase. Other features of this transcription unit include a high-efficiency eukaryotic translation start codon, a phage f1 origin of DNA replication for site-directed mutagenesis and a three-frame stop codon that facilitates C-terminal deletion mutagenesis. We term this vector family, pEXPRESS.