RNA Polymerase Core Enzyme Research Articles

Highly specific aptamer trap for extremophilic RNA polymerases

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus phylum. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ββ'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

Sigma factors of RNA polymerase in the pathogenic spirochaete Leptospira interrogans, the causative agent of leptospirosis.

The aim of this review is to summarize the current knowledge on the role of σ factors in a highly invasive spirochaete Leptospira interrogans responsible for leptospirosis that affects many mammals, including humans. This disease has a significant impact on public health and the economy worldwide. In bacteria, σ factors are the key regulators of gene expression at the transcriptional level and therefore play an important role in bacterial adaptative response to different environmental stimuli. These factors form a holoenzyme with the RNA polymerase core enzyme and then direct it to specific promoters, which results in turning on selected genes. Most bacteria possess several different σ factors that enable them to maintain basal gene expression, as well as to regulate gene expression in response to specific environmental signals. Recent comparative genomics and in silico genome-wide analyses have revealed that the L. interrogans genome, consisting of two circular chromosomes, encodes a total of 14 σ factors. Among them, there is one putative housekeeping σ70 -like factor, and three types of alternative σ factors, i.e., one σ54 , one σ28 and 11 putative ECF (extracytoplasmic function) σE -type factors. Here, characteristics of these putative σ factors and their possible role in the L. interrogans gene regulation (especially in this pathogen's adaptive response to various environmental conditions, an important determinant of leptospiral virulence), are presented.

Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE).

The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5'-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates β-Glucanase Expression in Tannerella forsythia in Response to Fusobacterium nucleatum Sensing.

The periodontal pathogen Tannerella forsythia expresses a β-glucanase (TfGlcA) whose expression is induced in response to Fusobacterium nucleatum, a bridge bacterium of the oral cavity. TfGlcA cleaves β-glucans to release glucose, which can serve as a carbon source for F. nucleatum and other cohabiting organisms. A two-gene cluster encoding a putative extracytoplasmic function (ECF) sigma factor and a FecR-like anti-sigma factor has been recognized upstream of a TfGlcA operon. We characterized and analyzed the role of these putative ECF sigma and anti-sigma factors in the regulation of TfGlcA expression. For this purpose, deletion mutants were constructed and analyzed for β-glucanase expression. In addition, an Escherichia coli-produced ECF sigma factor recombinant protein was evaluated for transcriptional and DNA binding activities. The results showed that the recombinant protein promoted transcription by the RNA polymerase core enzyme from the glcA promoter. Furthermore, in comparison to those in the parental strain, the β-glucanase expression levels were significantly reduced in the ECF sigma-factor deletion mutant and increased significantly in the FecR anti-sigma factor deletion mutant. The levels did not change in the mutants following coincubation with the F. nucleatum whole cells or cell extracts. Finally, the levels of β-glucanase produced by T. forsythia strains paralleled F. nucleatum biomass in cobiofilms. In conclusion, we identified a β-glucanase operon regulatory system in T. forsythia comprising an ECF sigma factor (TfSigG) and a cognate FecR-like anti-sigma factor responsive to F. nucleatum and potentially other stimuli. IMPORTANCE Previous studies have shown that F. nucleatum forms robust biofilms with T. forsythia utilizing glucose from the hydrolysis of β-glucans by T. forsythia β-glucanase, induced by F. nucleatum. In this study, we showed that a regulatory system comprising of an ECF sigma factor, TfSigG, and a FecR-like anti-sigma factor, TfFecR, is responsible for the β-glucanase induction in response to F. nucleatum, suggesting that this system plays roles in the mutualistic interactions of T. forsythia and F. nucleatum. The findings suggest the development and potential utility of small-molecule inhibitors targeting the β-glucanase activity or the TfSigG/TfFecR system as therapeutic drugs against dental plaque formation and periodontitis.

Rapid Adaptation Often Occurs through Mutations to the Most Highly Conserved Positions of the RNA Polymerase Core Enzyme.

Mutations to the genes encoding the RNA polymerase core enzyme (RNAPC) and additional housekeeping regulatory genes were found to be involved in adaptation, in the context of numerous evolutionary experiments, in which bacteria were exposed to diverse selective pressures. This provides a conundrum, as the housekeeping genes that were so often mutated in response to these diverse selective pressures tend to be among the genes that are most conserved in their sequences across the bacterial phylogeny. In order to further examine this apparent discrepancy, we characterized the precise positions of the RNAPC involved in adaptation to a large variety of selective pressures. We found that RNAPC lab adaptations tended to occur at positions displaying traits associated with higher selective constraint. Specifically, compared to other RNAPC positions, positions involved in adaptation tended to be more conserved in their sequences within bacteria, were more often located within defined protein domains, and were located closer to the complex’s active site. Higher sequence conservation was also found for resource exhaustion adaptations occurring within additional housekeeping genes. Combined, our results demonstrate that the positions that change most readily in response to well-defined selective pressures exerted in lab environments are often also those that evolve most slowly in nature.

The antiactivator FleN uses an allosteric mechanism to regulate σ54-dependent expression of flagellar genes in Pseudomonas aeruginosa.

Diverse sigma factors associate with the RNA polymerase (RNAP) core enzyme to initiate transcription of specific target genes in bacteria. σ54-Mediated transcription uses AAA+ activators that utilize their ATPase activity for transcription initiation. FleQ is a σ54-dependent master transcriptional regulator of flagellar genes in Pseudomonas aeruginosa. The ATPase activity of FleQ is regulated via a P-loop ATPase, FleN, through protein-protein interaction. We report a high-resolution crystal structure of the AAA+ domain of FleQ in complex with antiactivator FleN. The data reveal that FleN allosterically prevents ATP binding to FleQ. Furthermore, FleN remodels the region of FleQ essential for engagement with σ54 for transcription initiation. Disruption of the conserved protein-protein interface, by mutation, shows motility and transcription defects in vivo and multiflagellate phenotype. Our study provides a detailed mechanism used by monoflagellate bacteria to fine-tune the expression of flagellar genes to form and maintain a single flagellum.

Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and Anti-silencing by global regulators.

Transcriptional regulation for genome expression determines growth and adaptation of single-cell bacteria that are directly exposed to environment. The transcriptional apparatus in Escherichia coli K-12 is composed of RNA polymerase core enzyme and two groups of its regulatory proteins, seven species of promoter-recognition subunit sigma and about 300 species of transcription factors. The identification of regulatory targets for all these regulatory proteins is critical toward understanding the genome regulation as a whole. For this purpose, we performed a systematic search in vitro of the whole set of binding sites for each factor by gSELEX system. This review summarizes the accumulated knowledge of regulatory targets for more than 150 TFs from E. coli K-12. Overall TFs could be classified into four families: nucleoid-associated bifunctional TFs; global regulators; local regulators; and single-target regulators, in which the regulatory functions remain uncharacterized for the nucleoid-associated TFs. Here we overview the regulatory targets of two nucleoid-associated TFs, H-NS and its paralog StpA, both together playing the silencing role of a set of non-essential genes. Participation of LeuO and other global regulators have been indicated for the anti-silencing. Finally, we propose the hierarchy of TF network as a key framework of the bacterial genome regulation.

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein.

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World.

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea.

Biochemical and structural analyses reveal critical residues in δ subunit affecting its bindings to β′ subunit of Staphylococcus aureus RNA polymerase

A large class of bacterial RNA polymerase (RNAP) from low-G + C-content Gram-positive bacterial strains, such as the major human pathogen Staphylococcus aureus, not only contain five conserved subunits (αI, αII, β, β′ and ω), but also has a δ subunit. Despite being first identified as unique, Gram-positive specific component of RNAP apoenzyme more than 30 years ago and reported to be essential for transcription, the structural basis and molecular mechanism of δ subunit in the regulation of transcription remain poorly understood. Here, we performed structural analyses, site-directed mutagenesis and biochemical assays to uncover the interactions of S. aureus δ subunit with RNAP core enzyme and DNA towards the understanding of its role in transcription regulation. Microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA) of the wild-type and mutated S. aureus δ subunit revealed the N-terminal domain of δ subunit directly binds to the β′ jaw of S. aureus RNAP (SauRNAP), identified the key amino acid residues (F58, D61, D65, R67 and W81) of δ subunit involving in the binding with SauRNAP core enzyme, and uncovered the δ subunit C-terminal domain interferes with the interaction between DNA and SauRNAP core enzyme, by which transcription is regulated. Our results provide an excellent starting point for understanding the unique regulatory role and physiological function of δ subunit on transcription regulation in Gram-positive bacteria.

Resonance assignments of the cytoplasmic domain of ECF sigma factor W pathway protein YsdB from Bacillus subtilis.

Bacterial sigma (σ) factor, along with RNA polymerase core enzyme, initiates gene transcription from specific promoter regions and therefore regulates clusters of genes in response to a particular situation. The extracytoplasmic function (ECF) σ factors are a class of alternative σ factors that are often associated with environmental signal transduction across the bacterial membrane, in which external signal triggers the release of active σ from the membrane-anchored anti-σ factor. Gram-positive model organism Bacillus subtilis (B. subtilis) has seven ECF σ factors: σM, σV, σX, σW, σY, σZ and σYlaC. Although all these ECF σ factors were found to be involved in B. subtilis antibiotic resistance, σW is among the most studied and considered to play a pivotal role in responding to antimicrobial stresses. σW is under tight control and remains deactivated until exposure to external stimuli, after which proteases PrsW and RasP cleave the specific anti-sigma factor-RsiW to release and activate σW. Membrane anchored protein YsdB is a negative regulator of this activation, possibly via its direct interaction with PrsW and/or RsiW. Importantly, YsdB is well conserved among Bacilli, including pathogenic bacteria like Bacillus cereus. In this study, we describe the chemical shift assignments of the cytoplasmic domain of YsdB (29-130) of B. subtilis in solution as a basis for further interaction studies and structure determination. The near-complete assignment and the solution structure that will follow could provide a further understanding in σW regulation.

The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription

The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αββ‘ωε) RNA Polymerase (RNAP) core enzyme, σA, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the −35 element to the −10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal −35/−10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs.

Gene expression profiling of chromosome 10 in PTEN-knockout (−/−) human neural and mesenchymal stem cells: A system biology study

The present study investigates the effects of PTEN deletion in human neuro and mesenchymal stem cells related to the chromosome 10 gene expression profile. The RNA sequencing (RNA-seq) performed on four neural and four mesenchymal stem cells. DEG analysis outcome revealed 122 genes for neural stem cells (57 up-regulated and 65 down-regulated genes) and 258 genes for mesenchymal stem cells (98 up-regulated and 160 down-regulated genes) that were deferentially expressed in the PTEN (-/-) group compared to the normal group. Gene ontology analysis indicated that in the NSCs upregulated DEGs were significantly enriched in transcriptional activator activity, phosphatidylinositol phosphate phosphatase activity, MAP kinase activity while the downregulated DEGs were mainly involved in glycolytic process through glucose-6-phosphate, canonical glycolysis, glucose catabolic process to pyruvate. Meanwhile, in MSCs, the upregulated DEGs were mainly enriched in positive regulation of cell migration, chromatin silencing complex, RNA polymerase core enzyme binding while the downregulated DEGs phosphofructokinase activity, focal adhesion, glycolytic process through glucose-6-phosphate. On the basis of the PPI network of the DEGs, the following top hub genes were detected: six hub genes (PTEN, MAPK8, VCL, PAX2, ITGB1 and RET) in neural stem cells and 9 hub genes (PTEN, CDK1, KIF11, VCL, HK1, KIF20B, SIRT1, ACTR1A and ITGB1) appeared in mesenchymal stem cells respectively in each of the top 10 gene lists. In conclusion, PTEN, VCL and ITGB are found to be the main hub genes in both neural and mesenchymal stem cells, which involve in cell cycle, tumor progression and metastasis.

Adaptations Accumulated under Prolonged Resource Exhaustion Are Highly Transient.

Many nonsporulating bacterial species can survive for years within exhausted growth media in a state termed long-term stationary phase (LTSP). We have been carrying out evolutionary experiments aimed at elucidating the dynamics of genetic adaptation under LTSP. We showed that Escherichia coli adapts to prolonged resource exhaustion through the highly convergent acquisition of mutations. In the most striking example of such convergent adaptation, we observed that across all independently evolving LTSP populations, over 90% of E. coli cells carry mutations to one of three specific sites of the RNA polymerase core enzyme (RNAPC). These LTSP adaptations reduce the ability of the cells carrying them to grow once fresh resources are again provided. Here, we examine how LTSP populations recover from costs associated with their adaptation once resources are again provided to them. We demonstrate that due to the ability of LTSP populations to maintain high levels of standing genetic variation during adaptation, costly adaptations are very rapidly purged from the population once they are provided with fresh resources. We further demonstrate that recovery from costs acquired during adaptation under LTSP occurs more rapidly than would be possible if LTSP adaptations had fixed during the time populations spent under resource exhaustion. Finally, we previously reported that under LTSP, some clones develop a mutator phenotype, greatly increasing their mutation accumulation rates. Here, we show that the mechanisms by which populations recover from costs associated with fixed adaptations may depend on mutator status.IMPORTANCE Many bacterial species can survive for decades under starvation, following the exhaustion of external growth resources. We have previously shown that bacteria genetically adapt under these conditions in a manner that reduces their ability to grow once resources again become available. Here, we study how populations that have been subject to very prolonged resource exhaustion recover from costs associated with their adaptation. We demonstrate that rapid adaptations acquired under prolonged starvation tend to be highly transient, rapidly reducing in frequency once bacteria are no longer starved. Our results shed light on the longer-term consequences of bacterial survival under prolonged starvation. More generally, these results may also be applicable to understanding longer-term consequences of rapid adaptation to additional conditions as well.

Structural Insights into Transcription Initiation from De Novo RNA Synthesis to Transitioning into Elongation.

SummaryIn bacteria, the dissociable σ subunit of the RNA polymerase (RNAP) is responsible for initiating RNA synthesis from specific DNA sites. As nascent RNA grows, downstream DNA unwinds and is pulled into the RNAP, causing stress accumulation and initiation complex destabilization. Processive transcription elongation requires at least partial separation of the σ factor from the RNAP core enzyme. Here, we present a series of transcription complexes captured between the early initiation and elongation phases via in-crystal RNA synthesis and cleavage. Crystal structures of these complexes indicate that stress accumulation during transcription initiation is not due to clashing of the growing nascent RNA with the σ3.2 loop, but results from scrunching of the template strand DNA that is contained inside the RNAP by the σ3 domain. Our results shed light on how scrunching of template-strand DNA drives both abortive initiation and σ-RNAP core separation to transition transcription from initiation to elongation.

Role of Interactions of the CRE Region of Escherichia coli RNA Polymerase with Nontemplate DNA during Promoter Escape.

RNA polymerase (RNAP) recognizes promoter DNA through many interactions that determine specificity of transcription initiation. In addition to the dedicated transcription initiation σ factor in bacteria, the core enzyme of RNAP can also participate in promoter recognition. In particular, guanine residue at the+2 position (+2G) of the nontemplate DNA strand is bound in the CRE pocket formed by the RNAP β subunit. Here, we analyzed the role of these contacts in the process of promoter escape by RNAP by studying point mutations in the β subunit of Escherichia coli RNAP that disrupted these interactions. We found that the presence of+2G in the promoter slowed down the rate of promoter escape and increased proportion of inactive complexes. Amino acid substitutions in the CRE pocket decreased the promoter complex stability and changed the pattern of short RNA products synthesized during initiation, but did not significantly affect the rate of transition to elongation, regardless of the presence of+2G. Thus, the contacts of the CRE pocket with+2G do not make a significant contribution to the kinetics of promoter escape by RNAP, while the observed changes in the efficiency of abortive synthesis are not directly related to the rate of promoter escape.

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp0 strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp0 strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp0 suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp0 backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.

Coordinated Regulation of Rsd and RMF for Simultaneous Hibernation of Transcription Apparatus and Translation Machinery in Stationary-Phase Escherichia coli.

Transcription and translation in growing phase of Escherichia coli, the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS. The anti-sigma factor Rsd participates for the efficient replacement of sigma, and the unused RpoD is stored silent as Rsd–RpoD complex. On the other hand, functional 70S ribosome is transformed into inactive 100S dimer by two regulators, ribosome modulation factor (RMF) and hibernation promoting factor (HPF). In this review article, we overview how we found these factors and what we know about the molecular mechanisms for silencing transcription apparatus and translation machinery by these factors. In addition, we provide our recent findings of promoter-specific transcription factor (PS-TF) screening of the transcription factors involved in regulation of the rsd and rmf genes. Results altogether indicate the coordinated regulation of Rsd and RMF for simultaneous hibernation of transcription apparatus and translation machinery.