Rrn Promoter Research Articles

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate.

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus-E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source.

Kinetic properties of rrn promoters in Escherichia coli

How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter- lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [ R f], and the ratio of the Michaelis-Menten parameters, V max/ K m (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD 460) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD 460 and corresponds to an intracellular concentration of about 50 μM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.

Regulation of Sinorhizobium meliloti 1021 rrnA-reporter gene fusions in response to cold shock.

We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic (> or = 28-fold) increase in luminescence following a temperature downshift from 30 to 15 degrees C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrn-luxAB transcripts into active protein at 30 degrees C. This effect, however, was largely eliminated at 15 degrees C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.

Regulation of plastid rDNA transcription by interaction of CDF2 with two different RNA polymerases

The plastid genome is known to be transcribed by a plastid-encoded prokaryotic-type RNA polymerase (PEP) and by a nucleus-encoded phage-type RNA polymerase (NEP). The spinach plastid rrn operon promoter region harbours three different, overlapping promoters. Two of them are of the prokaryotic type. The third promoter is a non-consensus-type NEP promoter. We separated three different transcriptional activities from spinach chloroplasts: PEP, the phage-type RNA polymerase NEP-1, and a third, hitherto undescribed transcriptional activity (NEP-2). NEP-2 specifically transcribes the rrn operon in the presence of the transcription factor CDF2. CDF2 was previously shown to recruit PEP to the rrn promoter to repress transcription. Together, our results suggest the existence of a third RNA polymerase in plastids and a mechanism of rDNA transcriptional regulation that is based on the interaction of the transcription factor CDF2 with two different transcriptional systems.

Transcription from heterologous rRNA operon promoters in chloroplasts reveals requirement for specific activating factors.

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a sigma70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.

Elevated Fis expression enhances recombinant protein production in Escherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138–144, 1997.

Elevated Fis expression enhances recombinant protein production inEscherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.

Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum.

By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma80, was cloned and its nucleotide sequence was established. The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity). Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA polymerase core enzyme. A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserved regions 1 and 2, which might function as an interdomain linker. We purified the B. japonicum RNA polymerase holoenzyme. One of the subunits had an apparent molecular mass of 90 kDa and corresponded to the sigA gene product, as judged by N-terminal amino acid sequencing. The purified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rrn and groESL4 operons. They were identical to those previously identified in vivo. The rrn promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases. This served as a suitable template to analyze promoter activity. Then mutant derivatives of the rrn promoter were constructed and tested in in vitro transcription experiments. Several base pairs essential for promoter activity were thus identified. The results suggest that the well-characterized -35/-10 promoter class is predominantly used in B. japonicum for the expression of "housekeeping" genes.

Physiological and genetic regulation of rRNA synthesis in Lactococcus.

The macromolecular composition of Lactococcus was regulated by growth rate in the same general way as that of less fastidious bacteria such as Escherichia coli and Salmonella typhimurium. The ratios of RNA:DNA and RNA:protein increased approximately threefold over a 13.5-fold increase in growth rate, whereas the ratio of DNA:protein remained approximately constant. Using reporter genes fused to a DNA fragment of a cloned lactococcal rRNA operon, promoter activity was located upstream of the 16S rRNA structural gene. This DNA fragment had some characteristics typical of a rrn promoter in E. coli. Two consensus promoter sequences P1 and P2 were located 296 and 157 bp, respectively, upstream of the start of the 16S rRNA gene. Between P2 and the start of the 16S rRNA gene, sequences were identified with typical anti-termination motifs characteristic of E. coli rrn promoter regions. A putative transcription terminator sequence was identified downstream of the 5S rRNA gene and putative primary RNA transcript processing sites at both ends of the lactococcal rRNA operon were also noted.

Mechanisms of upstream activation of the rrnD promoter P1 of Escherichia coli

The rrn promoter regions of Escherichia coli contain a stretch of DNA, rich in An and Tn homopolymer sequences, which is located upstream of the tandem promoters P1 and P2. We have studied the effects of the upstream sequence of the rrnD operon on promoter function, using deletion variants for in vitro transcription. The presence of the upstream activating sequence (UAS) was found to increase P1 promoter strength without influencing P2. Two modes of P1 activation could be distinguished: a stimulation of P1 depending on the interaction of the factor of inversion stimulation (FIS) with the UAS (within the deletion boundaries of -50 and -112) and second, a factor-independent activation requiring the proximal part of the UAS (within the boundaries of -50 and -89). Both modes of activation were previously observed in the case of the rrnB operon and were ascribed to increased constants of RNA polymerase binding to P1 (Leirmo, S., and Gourse, R. L. (1991) J. Mol. Biol. 220, 555-568; Zacharias, M., Theissen, G., Bradaczek, C., and Wagner, R. (1991) Biochimie 73, 699-712). Our results show, however, that the mechanisms of upstream activation may vary with the reaction conditions. In a complex transcription system, originally designed for the use of cell extracts, FIS enhances first-order reactions that convert binary (open) complexes to transcribing complexes. Initiated complexes are stabilized by FIS. The factor-independent mode of P1 activation involves influences of the UAS on complex isomerizations as well as on binary complex formation. The results show that low molecular weight components of the complex transcription system change the function of the RNA polymerase at the rrn promoters (not at the reference promoter Ptac), so that the conversion of open to transcribing P1-complexes becomes dependent on the UAS and FIS.

Functional characteristics of the rrnD promoters of Escherichia coli.

The function of the tandem rrnD promoters (P1, P2) of Escherichia coli, which are highly efficient in directing rRNA synthesis, was studied in vitro using the strong hybrid promoter PtacI as a reference. One of the characteristics of the rrnD promoters is a pronounced instability of binary and initiating complexes formed with RNA polymerase. The rate of productive complex formation and of chain initiation at these promoters was found to be limited by a step in binary complex transitions with an apparent first-order rate constant equal to 3.9 x 10(-2) s-1. A comparison of this rate with that determined previously by filter binding assays (Gourse, R. (1988) Nucleic Acids Res. 16, 9789-9809) suggests that the rate-limiting step is a conversion of an intermediate species of open complex to one that is efficient in productive initiation. The slow rate of this reaction and the instability of open complexes account for the relatively low competitive strengths of the rrnD promoters. However, this limitation of rrn promoter function changes with promoter occupancy because the rate of chain initiation increased after completion of the first round of initiation. Despite their poor competitive strength, the rrnD promoters are more productive than PtacI at nonlimiting RNA polymerase concentrations. This can be ascribed to the different rates with which RNA polymerases leave PtacI and the rrnD promoters. These functional differences of the promoters are consistent with a "stressed intermediate" model of chain initiation (Straney, D.C., and Crothers, D.M. (1987) J. Mol. Biol. 193, 267-278) which predicts that rapid clearance of the rrn promoters is mechanistically related to the instability of the binary complexes.

Differential transcriptional control of the two tRNA fMet genes of Escherichia coli K-12

The metZ gene of Escherichia coli, which encodes the tRNA f Met, was cloned. Using the nucleotide sequence, in vitro transcription, and S 1 nuclease mapping analyses, we identified the promoter region, transcriptional start point, the two tandem tRNA f Met structural genes separated by an intergenic space of 33 bp, and the two Rho-independent transcriptional termination sites, in that order. We compared the promoter region of the metZ gene with that of the metY gene, which encodes the tRNA f2 Met and is located in the promoter-proximal portion of the nusA operon. A G + C-rich sequence (5′-GCGCATCCAC-3′), similar to the corresponding sequence of the rrn promoters that are under stringent control, was found between the Pribnow box and the transcriptional start point of the metZ promoter, but not in the metY promoter region. We therefore examined the effect of guanosine 3′-diphosphate, 5′-diphosphate (ppGpp), the chemical mediator of stringent control, and found that ppGpp inhibited the transcription of the metZ gene, but not that of the metY gene. These data suggested that the promoters for metZ and metY have different physiological functions and are regulated by different mechanisms.

Analysis of the promoter region in the rrnA operon from a blue-green alga, Anacystis nidulans 6301

The nucleotide sequence has been determined of a 1431 base pair (bp) region upstream from the 16S rRNA gene in the rrnA operon of a blue-green alga, Anacystis nidulans 6301. The transcriptional initiation site has been identified by S1 mapping with in vivo RNAs and in vitro transcripts synthesized by A. nidulans RNA polymerase. The initiation site is located 186 bp upstream from the 16S rRNA coding region both in vivo and in vitro. Escherichia coli RNA polymerase can recognize the authentic initiation site in vitro. The rrn promoter sequence of A. nidulans has more similarities with that of chloroplasts than that of E. coli. A large stem and loop structure can be constructed between 5′ and 3′ flanking sequences of the 16S rRNA. Three open reading frames (ORF) were found upstream from the rrnA promoter on the opposite strand, two of which are overlapped by 70 bp.

E coli RNA polymerase-rRNA promoter interaction and the effect of ppGpp.

The interaction between RNA polymerase and the E coli r (ibosomal) RNA promoters of the rrnX and rrnE operon was studied with the filter-binding technique. Quantitative differences were observed between the rrnX and rrnE promoters: stable rrnX promoter complexes are formed faster, and are less sensitive towards heparin and salt than stable rrnE promoter complexes. The effect of ppGpp, the specific inhibitor of rRNA synthesis, on rrn promoter complex formation was studied. In the presence of ppGpp complexes are formed which cannot be trapped in a transcription complex by addition of the start nucleotides, and are therefore considered to be non-productive. A tentative mode for the action of ppGpp is proposed.