Reconstituted RNA Polymerase Research Articles

Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts.

Genomic analyses reveal that RNA polymerase II initiates transcription but pauses shortly downstream on thousands of promoters in Drosophila and mammalian cells. Here, we describe the reconstitution of this promoter proximal pausing in nuclear extracts from Drosophila embryos. This approach is useful for dissecting the role(s) of transcription factors in promoter proximal pausing. Most of our studies employ the hsp70 heat shock gene promoter; however, this technique has successfully reconstituted RNA polymerase II pausing downstream of several other Drosophila promoters. A pulse/chase method is employed to restrict incorporation of radiolabel to the 5' portion of the RNA such that the specific activity of most transcripts are nearly identical and the intensity of radioactive RNA bands detected on gels reflects the molar ratios and quantities of each RNA product, regardless of length. The radiolabeled RNAs are isolated by hybridization to a biotinylated oligonucleotide and captured on magnetic beads. We also describe the use of antibodies to investigate mechanistic aspects of promoter proximal pausing.

The cyanobacterial principal σ factor region 1.1 is involved in DNA-binding in the free form and in transcription activity as holoenzyme

Cyanobacterial principal σ factor, σ A, includes a specifically conserved cluster of basic amino acids in the amino-terminal extension called region 1.1. We found that the σ A in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 binds DNA in the absence of the core RNA polymerase and that σ A lacking region 1.1 is not able to bind DNA. This indicates that, in the cyanobacterium, region 1.1 participates in DNA-binding, rather than inhibiting the interaction between free σ and DNA, as found in other principal σ factors of eubacteria. The results of in vitro transcription assays with the reconstituted RNA polymerase showed that region 1.1 reduces transcription activity from the cpc promoter.

藍藻 PCC 6803株における再構成 RNA ポリメラーゼを用いた in vitro 転写実験系の確立とシグマ因子のプロモーター認識能解析

藍藻 PCC 6803株における再構成 RNA ポリメラーゼを用いた in vitro 転写実験系の確立とシグマ因子のプロモーター認識能解析

Role of 5′-TGN-3′ motif in the interaction of mycobacterial RNA polymerase with a promoter of ‘extended −10’ class

In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the ‘extended −10’ class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by σ A, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position −37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of >75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG − mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription.

Promoter Escape by RNA Polymerase II: DOWNSTREAM PROMOTER DNA IS REQUIRED DURING MULTIPLE STEPS OF EARLY TRANSCRIPTION

Recent evidence, obtained in a reconstituted RNA polymerase II transcription system, indicated that the promoter escape stage of transcription requires template DNA located downstream of the elongating polymerase. In the absence of downstream DNA, very early elongation complexes are unable to synthesize transcripts longer than approximately 10-14 nucleotides. In contrast, once transcripts longer than approximately 15 nucleotides have been synthesized, an extended region of downstream DNA is no longer required (Dvir, A., Tan, S., Conaway, J. W., and Conaway, R. C. (1997) J. Biol. Chem. 272, 28175-28178). In this work, we sought to define precisely when, during the synthesis of the first 10-15 phosphodiester bonds, downstream DNA is required. We report that, for complete promoter escape, downstream DNA extending to position 40/42 is required. The polymerase can be forced to arrest at several points prior to the completion of promoter escape by removing downstream DNA proximally to positions 40/42. The positions at which the polymerase arrests appear to be determined by the length of available downstream DNA, with arrest occurring at a relatively fixed position of approximately 28 nucleotides to the distal end of the template. A similar requirement is observed for transcription initiation, i.e. the formation of the first phosphodiester bond of nascent transcripts. In addition, we show that the requirement for a downstream region is independent of downstream DNA sequence, suggesting that the requirement reflects a general mechanism. Taken together, our results indicate (i) that downstream DNA is required continuously through the synthesis of the first 14-15 phosphodiester bonds of nascent transcripts, and (ii) that a major conformational change in the transcription complex likely occurs only after the completion of promoter escape.

Regulation of sigma factor competition by the alarmone ppGpp.

Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

Transcription analysis of rpoH in Pseudomonas putida.

We previously determined the complete DNA sequence of the rpoH gene encoding the heat-shock sigma factor (sigmaH) of Pseudomonas putida. In the present study, the transcriptional start sites of rpoH were determined to be 41 nucleotides (T1), 153 nucleotides (T2) and 157 nucleotides (T3) upstream from the translational start codon (AUG) of rpoH by rapid amplification of cDNA 5'-ends. Based on the locations of T2 and T3, a sigma70-type promoter (P2) was determined to be located in the open reading frame region of upstream ftsX in addition to the sigmaE-type promoter (P1; DNA Res. 6 (1999) 241). In the in vitro transcription assay with reconstituted RNA polymerases (Esigma70, EsigmaE, EsigmaH and EsigmaS) of Pseudomonas aeruginosa, EsigmaE transcribed rpoH from T1 and Esigma(70) transcribed it from T2 and T3. In both cases, the level of transcription was higher at 42 degrees C than at 30 degrees C. No transcript was detected when EsigmaH or EsigmaS was used. These results indicate that EsigmaE and Esigma70 recognize P1 promoter and P2 promoter, respectively, and also prove that the synthesis of rpoH mRNA is inducible upon heat shock.

Transcription analysis of rpoH in Pseudomonas putida

We previously determined the complete DNA sequence of the rpoH gene encoding the heat-shock σ factor (σ H) of Pseudomonas putida. In the present study, the transcriptional start sites of rpoH were determined to be 41 nucleotides (T1), 153 nucleotides (T2) and 157 nucleotides (T3) upstream from the translational start codon (AUG) of rpoH by rapid amplification of cDNA 5′-ends. Based on the locations of T2 and T3, a σ 70-type promoter (P2) was determined to be located in the open reading frame region of upstream ftsX in addition to the σ E-type promoter (P1; DNA Res. 6 (1999) 241). In the in vitro transcription assay with reconstituted RNA polymerases (Eσ 70, Eσ E, Eσ H and Eσ S) of Pseudomonas aeruginosa, Eσ E transcribed rpoH from T1 and Eσ 70 transcribed it from T2 and T3. In both cases, the level of transcription was higher at 42°C than at 30°C. No transcript was detected when Eσ H or Eσ S was used. These results indicate that Eσ E and Eσ 70 recognize P1 promoter and P2 promoter, respectively, and also prove that the synthesis of rpoH mRNA is inducible upon heat shock.

Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription.

The human ISWI-containing factor RSF (remodeling and spacing factor) was found to mediate nucleosome deposition and, in the presence of ATP, generate regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation was impaired in directing transcription. Histone-tail modifications were found to regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 was found to increase transcription. Methylation of the histone H3 tail by Suv39H1 was found to repress transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system was refractory to the effect imposed by acetylation and methylation.

A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system.

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.

FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a −10 extended module but lacks its −35 hexamer; (ii) FruR binds to a target DNA region centered around position −45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120° in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in the C-terminal domain of their α subunit. The α[L262A], α[R265A] and α[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.

Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes.

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Esigma32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7. A point mutation at position -34 that is towards the E. coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Esigma38). (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10-19, 1998).

Assays for Investigating Transcription by RNA Polymerase IIin Vitro

With the availability of the general initiation factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), it is now possible to investigate aspects of the mechanism of eukaryotic messenger RNA synthesis in purified, reconstituted RNA polymerase II transcription systems. Rapid progress in these investigations has been spurred by use of a growing number of assays that are proving valuable not only for dissecting the molecular mechanisms of transcription initiation and elongation by RNA polymerase II, but also for identifying and purifying novel transcription factors that regulate polymerase activity. Here we describe a variety of these assays and discuss their utility in the analysis of transcription by RNA polymerase II.

The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo.

A general repressor extensively studied in vitro is the human Dr1/DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1 in vivo, the yeast Saccharomyces cerevisiae Dr1/DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated YDR1 and BUR6. The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1/Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation. Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies in vitro, we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription.

Transcription Activation by the Bacteriophage Mu Mor Protein Requires the C-terminal Regions of Both α and σ70 Subunits of Escherichia coli RNA Polymerase

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma70. As predicted if alpha were binding to Pm, we detected a polymerase-dependent footprint in the -60 region. Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from Pm in vitro. The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both alpha and sigma70; consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) alpha-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.

Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter. This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter. We have investigated which region of RNA polymerase contacts protein p4 at PA2c. Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription. Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c. Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.

Transcription factor recognition surface on the RNA polymerase alpha subunit is involved in contact with the DNA enhancer element.

The carboxy-terminal one-third of Escherichia coli RNA polymerase alpha subunit plays a key role in transcription regulation by a group of protein transcription factors and DNA enhancer (UP) elements. The roles of individual amino acid residues within this regulatory domain of the alpha subunit were examined after systematic mutagenesis of the putative contact regions (residues 258-275 and 297-298) for the cAMP receptor protein (CRP). The reconstituted RNA polymerases containing the mutant alpha subunits were examined for their response to transcription activation by cAMP-CRP and the rrnBP1 UP element. Mutations affecting CRP responsiveness were located on the surface of the putative CRP contact helix and most of these mutations also influenced the response to the rrnB UP element. These observations raise the possibility that the CRP contact surface is also involved in contact with the DNA UP element, although some amino acid residues within this region play different roles in molecular communication with CRP and the UP element. Among the amino acid residues constituting the contact surface, Arg265 was found to play a major role in response to both CRP and the DNA UP element. Judged by DNase I footprinting analysis, alpha mutants defective in transcription from the CRP-dependent lacP1 promoter showed decreased activity in the cooperative binding of CRP. Likewise, mutants defective in rrnBP1 transcription showed decreased binding to the UP element. The amino acid residues important for contact with both CRP and DNA are conserved in the alpha subunits of not only bacterial, but also chloroplast RNA polymerases.

Transcription activation by phage phi29 protein p4 is mediated by interaction with the alpha subunit of Bacillus subtilis RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage phi29 activates transcription from the viral late A3 promoter by stabilizing sigmaA-RNA polymerase at the promoter as a closed complex. Activation requires an interaction between protein p4 and RNA polymerase mediated by the protein p4 carboxyl-end, mainly through residue Arg-120. We have obtained derivatives of B. subtilis RNA polymerase alpha subunit with serial deletions at the carboxyl-end and reconstituted RNA polymerase holoenzymes harboring the mutant alpha subunits. Protein p4 promoted the binding of purified B. subtilis RNA polymerase alpha subunit to the A3 promoter in a cooperative way. Binding was abolished by deletion of the last 15 amino acids of the alpha subunit. Reconstituted RNA polymerases with deletions of 15 to 59 residues at the alpha subunit carboxyl-end could recognize and transcribe viral promoters not activated by protein p4, but they had lost their ability to recognize the A3 promoter in the presence of protein p4. In addition, these mutant reconstituted RNA polymerases could not interact with protein p4. We conclude that protein p4 activation of the viral A3 promoter requires an interaction between the carboxyl-end of protein p4 and the carboxyl-end of the alpha subunit of B. subtilis RNA polymerase that stabilizes the RNA polymerase at the promoter.

Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit.

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase alpha subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the alpha subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3' half of E. coli rpoA, the gene encoding the alpha subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the alpha subunit. Reconstituted RNA polymerases containing the mutant alpha subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the alpha subunit for OxyR.

The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature.

In vitro transcription analysis has shown that only RNA polymerase containing an alternative sigma subunit, sigma E, activates transcription from one of the rpoH promoters and the htrA promoter. The location of the rpoE gene encoding sigma E on the Escherichia coli chromosome has recently been established, but no rpoE mutant has yet become available for phenotypic testing. We cloned the rpoE gene from the lambda-ordered clones of the E. coli genome and confirmed that the reconstituted RNA polymerase containing the gene product (E sigma E) can transcribe htrA in vitro. We constructed an rpoE-defective strain by gene disruption using the cloned rpoE gene. We demonstrate that expression of htrA is completely dependent on the rpoE gene in vivo and that the rpoE gene is essential for bacterial growth at high temperature.