Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding.

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

Repression of deoP2 in Escherichia coli by CytR: conversion of a transcription activator into a repressor.

In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes. The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components. We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site. Interestingly, we also found that deoP2 carries an "UP-element" immediately upstream of the downstream cAMP-CRP site. The UP-element overlaps with the DNA site for CytR. However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the alpha-subunit as well as with intact RNA polymerase. The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of alpha, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2.

Novel mode of transcription regulation by SdiA, an Escherichia coli homologue of the quorum-sensing regulator.

SdiA, an Escherichia coli homologue of the quorum-sensing regulator, controls the expression of the ftsQAZ operon for cell division. Transcription of ftsQ is under the control of two promoters, upstream ftsQP2 and downstream ftsQP1, which are separated by 125 bp. SdiA activates transcription from ftsQP2 in vivo. Here, we demonstrate that SdiA facilitates the RNA polymerase binding to ftsQP2 and thereby stimulates transcription from P2. Gel shift and DNase I footprinting assays indicated that SdiA binds to the ftsQP2 promoter region between -51 and -25 with respect to the P2 promoter. Activation of ftsQP2 transcription by SdiA was observed with a mutant RNA polymerase containing a C-terminal domain (CTD)-deleted alpha-subunit (alpha 235) but not with RNA polymerase containing sigma(S) or a CTD-deleted sigma(D) (sigma(D)529). In good agreement with the transcription assay, no protection of P2 was observed with the RNA polymerase holoenzymes, E sigma(S) and E sigma(D)529. These observations together indicate that: (i) SdiA supports the RNA polymerase binding to ftsQP2; and (ii) this recruitment of RNA polymerase by SdiA depends on the presence of intact sigmaCTD. This is in contrast to the well-known mechanism of RNA polymerase recruitment by protein-protein contact between class I factors and alpha CTD. In addition to the P2 activation, SdiA inhibited RNA polymerase binding to the ftsQP1 promoter and thereby repressed transcription from P1. Gel shift assays indicate weak binding of SdiA to the P1 promoter region downstream from -13 (or +112 with respect to P2). Neither alpha CTD nor sigma CTD are required for this inhibition. Thus, the transcription repression of P1 by SdiA may result from its competition with the RNA polymerase in binding to this promoter.

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.

Functional analysis of RNA polymerase II Rpb3 mutants of the fission yeast Schizosaccharomyces pombe.

The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits. Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic alpha subunit, which plays a key role in RNA polymerase assembly. Together with the Rpb2 (the beta homologue) and Rpb11 (the second alpha homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2-Rpb3-Rpb11) which corresponds to the the alpha2beta assembly intermediate of prokaryotic RNA polymerase. For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S. pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3. The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S. pombe cell extracts. The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants. We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription.

Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.

Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase.

Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit. The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors.

Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit.

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface.

The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.

Regulatory sequences in sigma 54 localise near the start of DNA melting.

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

Two WD repeat-containing TATA-binding protein-associated factors in fission yeast that suppress defects in the anaphase-promoting complex.

The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4(+). The ubcP4(ts) mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome. One multicopy suppressor is the previously reported gene taf72(+), whereas the other is a previously unidentified gene named taf73(+). We show that the taf73(+) gene, like taf72(+), is essential for cell viability. The taf72(+) and taf73(+) genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9(+). These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis. Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle.

Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe.

The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase. To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S. pombe by quantitative immunoblotting. Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of beta', beta and alpha subunits, respectively, of prokaryotic RNA polymerase. On the other hand, the levels of small-sized subunits were between 2- to 15-fold higher than these three core subunits. The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II-specific core subunits. The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S. pombe whole cell extracts. The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms. Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S. pombe cell was estimated to be about 10,000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)-fused Rpb proteins. In agreement with the biochemical analysis, the GFP-Rpb1 and GFP-Rpb3 fusions were present in the nuclei but the GFP-Rpb4 was detected in the cytoplasm as well as the nuclei.

In vitro roles of invariant helix-turn-helix motif residue R383 in sigma(54) (sigma(N)).

In vitro DNA-binding and transcription properties of sigma(54) proteins with the invariant Arg383 in the putative helix-turn-helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme-promoter complexes in which limited DNA opening downstream of the -12 GC element has occurred. Unlike wild-type sigma(54), holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the -1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the sigma(54) holoenzyme, which includes the -12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of sigma(54).

Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression.

Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit. In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit. We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit. Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart. Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.

Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II.

The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III. At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits. To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay. The intracellular concentrations of the 12 Rpb subunits in growing S. pombe cells are different, within a range of 15-fold difference between the least abundant Rpb3 and the most abundant Rpb12. The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites. The promoters of the first group of genes contain the TATA box sequence between -26 and -62, while the second group of genes carry TATA-less promoters. Several common sequence segments, tentatively designated 'Rpb motifs', were identified in the promoter regions of the rpb genes. Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40-fold higher than the Rpb3 mRNA level. The levels of rpb mRNAs do not correlate with those of Rpb proteins. The protein-to-mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits beta', beta, alpha and alpha, respectively, of the prokaryotic RNA polymerase core enzyme.

Mode of DNA-protein interaction between the C-terminal domain of Escherichia coli RNA polymerase alpha subunit and T7D promoter UP element.

The C-terminal domain (CTD) downstream from residue 235 of Escherichia coli RNA polymerase alpha subunit is involved in recognition of the promoter UP element. Here we have demonstrated, by DNase I and hydroxyl radical mapping, the presence of two UP element subsites on the promoter D of phage T7, each located half and one-and-a-half helix turns, respectively, upstream from the promoter -35 element. This non-typical UP element retained its alphaCTD-binding capability when transferred into the genetic environment of the rrnBP1 basic promoter, leading to transcription stimulation as high as the typical rrnBP1 UP element. Chemical protease FeBABE conjugated to alphaCTD S309C efficiently attacked the T7D UP element but not the rrnBP1 UP element. After alanine scanning, most of the amino acid residues that were involved in rrnBP1 interaction were also found to be involved in T7D UP element recognition, but alanine substitution at three residues had the opposite effect on the transcription activation between rrnBP1 and T7D promoters. Mutation E286A stimulated T7D transcription but inhibited rrnBP1 RNA synthesis, while L290A and K304A stimulated transcription from rrnBP1 but not the T7D promoter. Taken together, we conclude that although the overall sets of amino acid residues responsible for interaction with the two UP elements overlap, the mode of alphaCTD interaction with T7D UP element is different from that with rrnBP1 UP element, involving different residues on helices III and IV.

Functional modulation of Escherichia coli RNA polymerase.

The promoter recognition specificity of Escherichia coli RNA polymerase is modulated by replacement of the sigma subunit in the first step and by interaction with transcription factors in the second step. The overall differentiated state of approximately 2000 molecules of the RNA polymerase in a single cell can be estimated after measurement of both the intracellular concentrations and the RNA polymerase-binding affinities for all seven species of the sigma subunit and 100-150 transcription factors. The anticipated impact from this line of systematic approach is that the prediction of the expression hierarchy of approximately 4000 genes on the E. coli genome can be estimated.

Mapping of functional domains on the influenza A virus RNA polymerase PB2 molecule using monoclonal antibodies.

Monoclonal antibodies against the PB2 of A/Puerto Rico/8/34 (A/PR/ 8/34) (H1N1) were prepared in order to define the functional domains of the RNA polymerase of influenza virus. The fifteen monoclonal antibodies that were generated were divided into 4 groups on the basis of ELISA binding to PB2 or its peptide fragments. Six Group I antibodies that bound to the PB2 N-terminal region (amino acids 1-104) did not inhibit transcription by the viral ribonucleoprotein complex. A single Group II antibody recognizing the region of amino acids 206-259 inhibited ApG-primed transcription. Groups III and IV antibodies bound to the C-terminal region of amino acids 660-759. Of these, Group III antibodies inhibited transcription. The present results identify multiple monoclonal antibody binding domains in PB2, two of which, when bound by antibodies, negatively affect viral RNA transcription.