Coli RNA Research Articles

Phosphopeptides derived from in vitro phosphorylated E. coli RNA polymerase bind to DNA and affect DNA transcription.

E. Coli RNA polymerase was phosphorylated with protein kinase CKII and allowed to bind to pBR322. After digestion of the RNA polymerase-pBR322 complex with proteinase K, the phosphopeptides that remained bound to DNA were extracted and analyzed. These phosphopeptides are able to bind again to DNA and to inhibit transcription.

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

Expression, Purification, and Characterization of an Active RNase H Domain of the Hepatitis B Viral Polymerase

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.

Structural Modules of the Large Subunits of RNA Polymerase: INTRODUCING ARCHAEBACTERIAL AND CHLOROPLAST SPLIT SITES IN THE β AND β′ SUBUNITS OF ESCHERICHIA COLI RNA POLYMERASE

The beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

Point mutations in a transcription terminator, λ tI, that affect both transcription termination and RNA stability

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage λ. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3′ exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81–93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8–12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tl is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3′ to 5′ exonucleolytic activity of polynucleotide phosphorylase.

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.

Molecular anatomy of the beta' subunit of the E. coli RNA polymerase: identification of regions involved in polymerase assembly.

The E. coli RNA polymerase is a multisubunit enzyme, which is present in two different forms: the catalytic competent core enzyme (alpha2beta beta') and the promoter selective holoenzyme (alpha2beta beta' sigma). Correct assembly of individual subunits into core or holoenzyme is essential for the function of this enzyme. Mutant beta' proteins truncated near the centre or at the C-terminus were able to form stable core enzyme-like complexes under reconstitution conditions. Mutant beta' proteins lacking the region between amino acids 201-477 failed to form holoenzyme complexes while retaining the ability to form core enzyme complexes. Furthermore, free beta' subunit interacted with free sigma subunit to form a stable beta' sigma subassembly. Removal of amino acids 201-477 from the beta' subunit strongly interfered with this interaction. Our results suggest that the N-terminal region of the beta' subunit is involved in the assembly of core enzyme. The region between amino acids 201 and 477 on beta' may be directly or indirectly involved in the interaction between the beta' subunit and the sigma subunit.

Function of E. coli RNA Polymerase σ Factor- σ 70 in Promoter-Proximal Pausing

The σ factor σ 70 of E. coli RNA polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage λ gene Q transcription antiterminator to act. We identify the signal in DNA that induces early pausing to be a version of the σ 70 −10 promoter consensus, and we show that σ 70 is both necessary for pausing and present in the paused transcription complex. Regions 2 and 3 of σ 70 suffice to induce pausing. Since pausing is induced by the nontemplate DNA strand of the open transcription bubble, we conclude that RNA polymerase containing σ 70 carries out base-specific recognition of the nontemplate strand as single stranded DNA. We suggest that σ 70 remains bound to core RNA polymerase when the −10 promoter contacts are broken, and then moves to the pause-inducing sequence.

Localization of nusA-suppressing amino acid substitutions in the conserved regions of the beta' subunit of Escherichia coli RNA polymerase.

Escherichia coli RNA polymerase is composed of four different subunits, alpha (present in two copies), beta, beta' and sigma. Among these, the beta' polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood. We isolated novel mutations in a plasmid-borne copy of rpoC, which encodes beta', as dominant suppressors of two temperature-sensitive nusA alleles. All 20 suppressors of nusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in the beta' subunit. In vivo and in vitro transcription assays revealed that the rpoC10 allele of beta' participates in Rho-dependent transcription termination. On the other hand, of 20 suppressors of nusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I. These results suggested that the conserved domains of the beta' subunit of E. coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).

Fluorescence spectroscopy analysis of active and regulatory sites of RNA polymerase

This chapter discusses the fluorescence spectroscopy analysis of active and regulatory sites of RNA polymerase. The chapter divides the primary method of fluorescence spectroscopy into two categories to understand both the functional and the structural aspects of Escherichia coli (E. Coli) RNA polymerase. First, it deals with the extrinsic probe labeling of the substrate and enzyme, and then discusses the different important conclusions that can be drawn from such studies. Next, the time-resolved emission spectroscopic analysis of tryptophan fluorescence and the structure-function relationship of a transcription factor are discussed. The chapter discusses the syntheses and characterization of (γ-AmNS) ATP and UTP. The labeling of E. coli RNA polymerase and its subunits is discussed. The E. coli RNA polymerase has five subunits that are essential for the catalytic functions of the enzyme. Most of the active site labeling of the enzyme has been carried out at the substrate level. The only subunit that has been studied, with the help of fluorescence spectroscopy in some detail, is the regulatory subunit of E. coli RNA polymerase or the subunit.

A Base Substitution within the GTPase-associated Domain of Mammalian 28 S Ribosomal RNA Causes High Thiostrepton Accessibility

A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA. Filter binding assay showed no detectable binding of the rat RNA to thiostrepton, but the binding capacity was markedly increased by base substitution of G1878 to A at the position corresponding to 1067 of Escherichia coli 23 S rRNA. The association constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was comparable with that of the E. coli RNA (K alpha = 1.1 x 10(6) M-1). This suggests that the eukaryotic G 1878 participates in the resistance for thiostrepton. On the other hand, the RNA fragments of the two species had a similar binding capacity for E. coli ribosomal protein L11 and its mammalian homologue L12. Gel electrophoresis under a high ionic condition, however, revealed a difference between the two proteins. E. coli L11 formed stable complexes with both the E. coli RNA and the rat A 1878 mutant RNA in the presence of thiostrepton, while rat L12 failed to exhibit such complex formation. This suggests that the eukaryotic L12 protein may also be an element giving the resistance for thiostrepton. These results are discussed in terms of preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eukaryotes.

Mutational analysis of the role of the first helix of region 4.2 of the sigma 70 subunit of Escherichia coli RNA polymerase in transcriptional activation by activator protein PhoB.

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70, which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif or region 4.2 of sigma 70. To study further the role of the first helix of the HTH motif of sigma 70 in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma 70 proteins. RNA polymerases containing the mutant sigma 70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma 70 (E574K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma 70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.

Effect of guanosine tetraphosphate (ppGpp) on the conformational state ofE. coli RNA polymerase and transcription directed by tyrT, T7D, and T7A1 promoters

Guanosine tetraphosphate (ppGpp) is an allosteric regulator of DNA-dependent RNA polymerase. It selectively inhibits transcription from some promoters containing the GC-rich sequence near the starting point of transcription. Using the method of the fluorescent label it was shown that interacting with the β-subunit of the enzyme ppGpp induces essential conformational transitions of the RNA polymerase. The mutation in the enzyme β-subunit that increases the polymerase affinity for T7D promoter affects ppGpp-induced conformational changes. The rate of the abortive RNA synthesis directed by the mutant enzyme from this promoter, which contains no GC-rich sequences near the starting point appeared to be ppGpp-dependent. ppGpp has no effect on T7D activity in the system containing the native enzyme. Thus, the regulatory function of ppGpp depends on the structure of the RNA polymerase and could be realized by the enzyme not only by recognition of the GC-rich block in the promoter.

Structural and functional analysis of T7D promoter and its complex withE. coli RNA polymerase

The DNA fragment containing D promoter of the T7 bacteriophage and its complex with DNA-dependent RNA polymerase undergo a conformational transition at 30 °C, which is accompanied by an increase in the rate of phosphodiester bond synthesis.

Analyses of the Extent of Immunological Relatedness between a Highly Purified Pea Chloroplast Functional RNA Polymerase and Escherichia coli RNA Polymerase

Summary The reported nucleotide sequence homology between the RNA polymerase (EC 2.7.7.6) coding (rpo) genes in Escherichia coli and their putative homologues in chloroplast DNA is not only limited to about 26-50 %, but also scattered. Utilising the homologous in vitro transcription system and immunological approach, we address as to which extent the chloroplast functional RNA polymerase and E. coli RNA polymerase are related. Highly purified RNA polymerase from pea chloroplasts has been demonstrated to transcribe both ribosomal and messenger RNA genes and the purified homologous antibodies inhibit the chloroplast gene promoter recognizing ability as well as in vitro transcriptional activity of the enzyme [Rajasekhar et al., 1991]. While recognizing all of the polypeptides of the chloroplast enzyme on Western blots, the chloroplast RNA polymerase antibodies did not cross-react with the sub-units of E. coli RNA polymerase or wheat germ RNA polymerase and vice versa. Antibodies against the 130 kDa,110 kDa, 75-95 kDa and 48 kDa polypeptides of the purified chloroplast RNA polymerase immobilized only homologous RNA polymerase on the affinity columns. The active enzyme eluted from the immunoaffinity columns showed sub-unit patterns similar to that of purified RNA polymerase. The specificity of these antibodies was confirmed with the lack of immobilization of E. coli RNA polymerase and vice versa with the E. coli RNA polymerase antibodies. The E. coli RNA polymerase also manifested transcription in vitro utilising the supercoiled 7A/30T recombinant containing the chloroplast 16S rRNA minigene as well as the supercoiled pCB 1-3 recombinant containing the upstream sequences of ribulose-1,5-bisphosphate carboxylase large sub-unit (rbc-L) gene. The transcriptional activities, however, were specifically affected only by homologous RNA polymerase antibodies, but not by heterologous RNA polymerase antibodies. While both the chloroplast and the E. coli RNA polymerases caused mobility shifts of the chloroplast 16S rRNA gene and rbc-L gene promoter fragments, only the homologous antibodies were effective in abolishing this response. The data obtained form the first functional evidence that the highly purified pea chloroplast RNA polymerase and the E. coli RNA polymerase are immunologically only distantly related.

Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex. Potential for gene-specific inactivation reagents

An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase. Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide. When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed. The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies.

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.

New Possibility for Metastable RNA Folding of Biological Significance: A Physico‐Chemical View at Biological Regulation and Control

AbstractWe assess the importance of short‐lived nonequilibrium RNA folding in biological regulation. As an illustration, we explain the radical difference in pause site patterns during the synthesis of MDV‐1 RNA, depending on the nature of the process involved. The two situations of interest are: a) Replication by Qß‐replicase, a template‐instructed synthesis, and b) Transcription performed by E. Coli RNA polymerase. We start by establishing the possibility of soliton‐induced tautomerization of uracil which occurs exclusively in transcription. This tautomeric form of uracil forms base‐pairing with guanine and leads to a unique metastable folding of RNA. The assessment of this fact enables us to prove that in both replication and transcription, the pauses in the process are due to refolding events occurring in the replica chain as it is being synthesized and in the upstream portion of the template strand.

Thermodynamic analysis of the transcription cycle in E. coli

The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.