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PubMed1996

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

ATP-Binding Cassette Transporters↗

PubMed1996

Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis.

The expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine. The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis. Whereas lys-C-specific RNA from lysine-starved B. subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript. On the other hand, a B. subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine. Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element. Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules. These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.

Bacillus subtilis↗

PubMed1999

Structure and organization of the rrnD operon of 'Brevibacterium lactofermentum': analysis of the 16S rRNA gene.

Five rRNA operons (rrn) were found by hybridization in the genome of 'Brevibacterium lactofermentum' ATCC 13869 and Corynebacterium glutamicum ATCC 13032. 'B. lactofermentum' DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-5S rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that 'B. lactofermentum' is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5' end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

Amino Acid Sequence↗

PubMed2003

An inducible tellurite-resistance operon in Proteus mirabilis.

Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.

Bacterial Proteins↗

PubMed2003

Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively.

Pseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene using the toluene degradation pathway, and can also utilize p-cymene via p-cumate using the p-cymene and p-cumate catabolic pathways. In the present study, P. putida F1 strains were isolated that were adapted to assimilate new substrates such as n-propylbenzene, n-butylbenzene, cumene and biphenyl, and the molecular mechanisms of genetic adaptation to an expanded range of aromatic hydrocarbons were determined. Nucleotide sequence analyses showed that the selected strains have mutations in the cymR gene but not in todF gene. The impairment of the repressor CymR by mutation led to the constitutive expression of CmtE, a meta-cleavage product hydrolase from the cmt operon. This study also showed that CmtE has a broad range of substrates and can hydrolyse meta-cleavage products formed from biphenyl and other new growth substrates via the toluene degradation pathway. However, the artificially constructed strain P. putida F1(cymR : : Tc(r)) and a recombinant P. putida F1, which expressed CmtE constitutively, could not grow on the new substrates. The adapted strains possess the tod operon, which is induced by new growth substrates that are poor inducers of wild-type P. putida F1. When the todS gene from the adapted strains was introduced in a trans manner to P. putida F1(cymR : : Tc(r)), the resulting recombinant strains were able to grow on biphenyl and other new substrates. This finding indicates that the TodS sensor was altered to recognize these substrates and this conclusion was confirmed by nucleotide sequence analyses. Amino acid substitutions were found in the regions corresponding to the receiver domain and the second PAS domain and their boundaries in the TodS protein. These results showed that P. putida F1 adapted strains capable of growth on n-propylbenzene, n-butylbenzene, cumene and biphenyl possess mutations to employ CmtE and to induce the tod catabolic operon by the new growth substrates.

Adaptation, Physiological↗

PubMed2003

Cloning and characterization of the groE heat-shock operon of the marine bacterium Vibrio harveyi.

The DNA region of the Vibrio harveyi chromosome containing the heat-shock genes groES and groEL was cloned, and the genes were sequenced. These genes are arranged in the chromosome in the order groES-groEL. Northern hybridization experiments with RNA from V. harveyi and a DNA probe carrying both groES and groEL genes showed a single, heat-inducible transcript of approximately 2200 nt, indicating that these genes form an operon. Primer extension analysis revealed a strong, heat-inducible transcription start site 59 nt upstream of groES, preceded by a sequence typical for the Escherichia coli heat-shock promoters recognized by the sigma(32) factor, and a weak transcription start site 25 nt upstream the groES gene, preceded by a sequence typical for sigma(70) promoters. Transcription from the latter promoter occurred only at low temperatures. The V. harveyi groE operon cloned in a plasmid in E. coli cells was transcribed in a sigma(32)-dependent manner; the transcript size and the sigma(32)-dependent transcription start site were as in V. harveyi cells. Comparison of V. harveyi groE transcription regulation with the other well-characterized groE operons of the gamma subdivision of proteobacteria (those of E. coli and Pseudomonas aeruginosa) indicates a high conservation of the transcriptional regulatory elements among these bacteria, with two promoters, sigma(32) and sigma(70), involved in the regulation. The ability of the cloned groESL genes to complement E. coli groE mutants was tested: V. harveyi groES restored a thermoresistant phenotype to groES bacteria and enabled lambda phage to grow in the mutant cells. V. harveyi groEL did not abolish thermosensitivity of groEL bacteria but it complemented the groEL mutant with respect to growth of lambda phage. The results suggest that the GroEL chaperone may be more species-specific than the GroES co-chaperone.

Base Sequence↗

PubMed2004

The transcription of the cbb operon in Nitrosomonas europaea.

Nitrosomonas europaea is an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles. N. europaea utilizes CO(2) as its predominant carbon source, and is an obligate chemolithotroph, deriving all the reductant required for energy and biosynthesis from the oxidation of ammonia (NH(3)) to nitrite (). This bacterium fixes carbon via the Calvin-Benson-Bassham (CBB) cycle via a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). The RubisCO operon is composed of five genes, cbbLSQON. This gene organization is similar to that of the operon for 'green-like' type I RubisCOs in other organisms. The cbbR gene encoding the putative regulatory protein for RubisCO transcription was identified upstream of cbbL. This study showed that transcription of cbb genes was upregulated when the carbon source was limited, while amo, hao and other energy-harvesting-related genes were downregulated. N. europaea responds to carbon limitation by prioritizing resources towards key components for carbon assimilation. Unlike the situation for amo genes, NH(3) was not required for the transcription of the cbb genes. All five cbb genes were only transcribed when an external energy source was provided. In actively growing cells, mRNAs from the five genes in the RubisCO operon were present at different levels, probably due to premature termination of transcription, rapid mRNA processing and mRNA degradation.

Bacterial Proteins↗

PubMed2004

The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex.

Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway.

Bacterial Proteins↗

PubMed2005

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons.

The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2.061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (approximately 0.05-4.3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.

Chromosome Mapping↗

PubMed2004

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence.

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

Animals↗

PubMed2005

Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ.

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

Bacterial Proteins↗

PubMed2005

Transcription and autoregulation of the Rv3134c-devR-devS operon of Mycobacterium tuberculosis.

DevR is a transcriptional regulator that mediates the genetic response of Mycobacterium tuberculosis to oxygen limitation and nitric oxide exposure. devR is co-transcribed along with devS, which encodes its cognate sensor kinase, and an upstream gene, Rv3134c. The transcriptional activity of this operon was characterized by primer extension, transcriptional fusion and electrophoretic mobility shift assays (EMSAs) under aerobic conditions. Transcription start points (Tsps) were detected upstream of both Rv3134c and devR, and the major transcript was derived from upstream of Rv3134c. Sequences with similarity to sigma factor consensus elements and to DevR-binding motifs were detected in the vicinity of the Tsps by in silico analysis. EMSAs with promoter regions and DevR protein showed that DevR binds to its own promoters in a sequence-specific manner with differing affinities. Consistent with the primer extension and EMSA data, Rv3134c promoters, and not devR promoters, were determined to be the principal promoters of this operon using reporter assays performed in Mycobacterium smegmatis and Escherichia coli. Furthermore, DevR modulated the activity of both devR and Rv3134c promoters. From these findings it is inferred that the Rv3134c-devR-devS operon is transcribed from multiple promoters and is autoregulated.

Bacterial Proteins↗

PubMed2006

Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon.

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

Bacterial Proteins↗

PubMed2002

Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon.

The physiological role of Escherichia coli Spot 42 RNA has remained obscure, even though the 109-nucleotide RNA was discovered almost three decades ago. Structural features of Spot 42 RNA and previous work suggested to us that the RNA might be a regulator of discoordinate gene expression of the galactose operon, a control that is only understood at the phenomenological level. The effects of controlled expression of Spot 42 RNA or deleting the gene (spf) encoding the RNA supported this hypothesis. Down-regulation of galK expression, the third gene in the gal operon, was only observed in the presence of Spot 42 RNA and required growth conditions that caused derepression of the spf gene. Subsequent biochemical studies showed that Spot 42 RNA specifically bound at the galK Shine-Dalgarno region of the galETKM mRNA, thereby blocking ribosome binding. We conclude that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit. Our results reveal an interesting mechanism by which the expression of a promoter distal gene in an operon can be modulated and underline the importance of antisense control in bacterial gene regulation.

Bacterial Proteins↗

PubMed1991

Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms.

The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the sigma-factor sigma 54. Negative regulation is mediated by both PspA and the sigma 32-dependent heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the sigma 32- and sigma E-dependent systems, for genes induced by heat and other stresses.

Bacterial Proteins↗

PubMed2005

Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation.

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

5' Untranslated Regions↗

PubMed2002

A two-component Mn2+-sensing system negatively regulates expression of the mntCAB operon in Synechocystis.

Mn is an essential component of the oxygen-evolving machinery of photosynthesis and is an essential cofactor of several important enzymes, such as Mn-superoxide dismutase and Mn-catalase. The availability of Mn in the environment varies, and little is known about the mechanisms for maintaining cytoplasmic Mn(2+) ion homeostasis. Using a DNA microarray, we screened knockout libraries of His kinases and response regulators of Synechocystis sp PCC 6803 to identify possible participants in this process. We identified a His kinase, ManS, which might sense the extracellular concentration of Mn(2+) ions, and a response regulator, ManR, which might regulate the expression of the mntCAB operon for the ABC-type transporter of Mn(2+) ions. Furthermore, analysis with the DNA microarray and by reverse transcription PCR suggested that ManS produces a signal that activates ManR, which represses the expression of the mntCAB operon. At low concentrations of Mn(2+) ions, ManS does not generate a signal, with resulting inactivation of ManR and subsequent expression of the mntCAB operon.

ATP-Binding Cassette Transporters↗

PubMed2003

Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants.

Expression of the plastid rRNA operon (rrn) during development is highly regulated at the level of transcription. The plastid rrn operon in most higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a sigma(70)-type promoter with conserved -10 and -35 core promoter elements. To identify functionally important sequences, the tobacco PrrnP1 was dissected in vivo and in vitro. Based on in vivo deletion analysis, sequences upstream of nucleotide -83 do not significantly contribute to promoter function. The in vitro analyses identified an essential hexameric sequence upstream of the -35 element (GTGGGA; the rRNA operon upstream activator [RUA]) that is conserved in monocot and dicot species and suggested that the -10 element plays only a limited role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, the characteristic of strong promoters in prokaryotes. We propose that sigma interaction with the -10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein-DNA) interaction or by protein-protein interaction between the PEP and an RUA binding transcription factor.

Base Sequence↗