Transcription Needs Translation Initiation of the Downstream Gene to Continue Downstream at Intercistronic Junctions in E. Coli.

We have reported a gal mutant called galE stop0, wherein the galE stop codon was changed to a sense codon. The experiment results demonstrated that preventing galE translation termination inhibited the production of galE 3' ends. This implies that when the galE translation termination was prevented, the galE 3' ends generation was reduced or impaired. We anticipated that the translation of galE would continue to galT, producing a chimeric protein GalE-GalT. This study verified that the chimeric protein was produced, but unexpectedly, we found that the GalT protein was also synthesized in the mutant, and its amount equaled that in the wild-type. In the wild-type, we also found that the GalE-GalT chimeric protein was produced in an amount equal to that of the GalE protein. These results suggest that translation termination of galE and translation initiation of galT occur independently, thus, corroborating our transcription-translation model: At the cistron junction, transcription, decoupled from translation due to the translation termination of galE, needs translation initiation of galT to continue downstream; otherwise, transcription would be terminated by Rho. RNase E-mediated transcript cleavage also produces the 3' ends of pre-galE mRNA. These findings indicated that RNase E produces the 3' end of mRNA and establishes gene expression polarity.

Reporter Gene-Based qRT-PCR Assay for Rho-Dependent Termination In Vivo.

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, (coding tRNAarg) from Brevibacterium albidum, and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNAarg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNAarg with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research.

sRNA expedites polycistronic mRNA decay in Escherichia coli.

In bacteria, most small RNA (sRNA) elicits RNase E-mediated target mRNA degradation by binding near the translation initiation site at the 5' end of the target mRNA. Spot 42 is an sRNA that binds in the middle of the gal operon near the translation initiation site of galK, the third gene of four, but it is not clear whether this binding causes degradation of gal mRNA. In this study, we measured the decay rate of gal mRNA using Northern blot and found that Spot 42 binding caused degradation of only a specific group of gal mRNA that shares their 3' end with full-length mRNA. The results showed that in the MG1655Δspf strain in which the Spot 42 gene was removed, the half-life of each gal mRNA in the group increased by about 200% compared to the wild type. Since these mRNA species are intermediate mRNA molecules created by the decay process of the full-length gal mRNA, these results suggest that sRNA accelerates the mRNA decaying processes that normally operate, thus revealing an unprecedented role of sRNA in mRNA biology.

Identification of a Rho-Dependent Termination Site In Vivo Using Synthetic Small RNA.

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3' end of mRNA is rapidly removed after RDT by 3'-to-5' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3' ends from rapid degradation by 3'-to-5' exonuclease, thereby stabilizing the mRNA 3' end. We further verified that the signal intensity at the 3' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.

Failure of Translation Initiation of the Next Gene Decouples Transcription at Intercistronic Sites and the Resultant mRNA Generation.

In Escherichia coli, transcription is coupled with translation. The polar gal operon is transcribed galE-galT-galK-galM; however, about 10% of transcription terminates at the end of galE because of Rho-dependent termination (RDT). When galE translation is complete, galT translation should begin immediately. It is unclear whether RDT at the end of galE is due to decoupling of translation termination and transcription at the cistron junction. In this study, we show that RDT at the galE/galT cistron junction is linked to the failure of translation initiation at the start of galT, rather than translation termination at the end of galE. We also show that transcription pauses 130 nucleotides downstream from the site of galE translation termination, and this pause is required for RDT. IMPORTANCE Transcription of operons is initiated at the promoter of the first gene in the operon, continues through cistron junctions, and terminates at the end of the operon, generating a full-length mRNA. Here, we show that Rho-dependent termination of transcription occurs stochastically at a cistron junction, generating a stable mRNA that is shorter than the full-length mRNA. We further show that stochastic failure in translation initiation of the next gene, rather than the failure of translation termination of the preceding gene, causes the Rho-dependent termination. Thus, stochastic failure in translation initiation at the cistron junction causes the promoter-proximal gene to be transcribed more than promoter-distal genes within the operon.

An in vitro Assay of mRNA 3' end Using the E. coli Cell-free Expression System.

At the end of about 80% of the operon in Escherichia coli, translation termination decouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3' end of the mRNA generated by RDT. Here, we present a cell-free assay system that could provide detailed information on the 3' end of a transcript RNA generated by RDT. Our protocol shows how to extract transcript RNA generated by transcription reactions from a cell-free extract, followed by an RNA oligomer ligation to the 3' end of a transcript RNA of interest. The 3' end of the RNA is amplified using RT-PCR. Its genetic location can be determined using a gene-specific primer extension reaction. The 3' ends of mRNA can be visualized and quantified by polyacrylamide gel electrophoresis. One significant advantage of a cell-free assay system is that factors involved in the generation of the 3' end, such as proteins and sRNA, can be directly assayed by exogenously adding factor(s) to the reaction. Graphic abstract: An illustration of the experimental methodology.

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3'-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic gal mRNA.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5'-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5'-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5' end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.

Erratum: Publisher Correction: A c-di-AMP riboswitch controlling kdpFABC operon transcription regulates the potassium transporter system in Bacillus thuringiensis.

[This corrects the article DOI: 10.1038/s42003-019-0414-6.].

A c-di-AMP riboswitch controlling kdpFABC operon transcription regulates the potassium transporter system in Bacillus thuringiensis.

The intracellular K+ level in bacteria is strictly controlled by K+ uptake and efflux systems. Among these, KdpFABC is a high-affinity K+ transporter system that is generally activated by the KdpDE two-component system in response to K+ limitation stress. However, the regulatory mechanism remains obscure in bacteria lacking the kdpDE genes. Here we report that the transcription of a kdpFABC operon is distinctively regulated by a cyclic diadenylate monophosphate (c-di-AMP) riboswitch located at the 5'-untranslated region of kdp transcript, and binding of c-di-AMP to the riboswitch promotes its intrinsic termination that blocks the kdpFABC transcription. Further, the intracellular c-di-AMP concentration was found to decrease under the K+ limitation stress, leading to transcriptional read-through over the terminator to allow kdpFABC expression. This regulatory element is found predominantly in the Bacillus cereus group and correlate well with the K+ and c-di-AMP homeostasis that affects a variety of crucial cellular functions.

Processing generates 3' ends of RNA masking transcription termination events in prokaryotes.

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation-transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3' ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3' RNA ends; thus the in vivo 3' ends do not define termination sites. We predict generation of 3' ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.

Visualization of RNA 3' ends in Escherichia coli Using 3' RACE Combined with Primer Extension.

In this assay, 3' RACE (Rapid Amplification of cDNA 3' Ends) followed by PE (primer extension), abbreviated as 3' RACE-PE is used to identify the mRNA 3' ends. The following protocol describes the amplification of the mRNA 3' ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5' end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3' RACE. The conventional PE can only be used to locate the 5' end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5' → 3' direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3' end of the mRNA using this assay. The relative amount of the 3' end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3' ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.

High-resolution crystal structure of the PDZ1 domain of human protein tyrosine phosphatase PTP-Bas.

Protein tyrosine phosphatase-Basophil (PTP-Bas) is a membrane-associated protein tyrosine phosphatase with five PDZ domains and is involved in apoptosis, tumorigenesis, and insulin signaling. The interaction between PTP-Bas and tandem-PH-domain-containing protein 1/2 (TAPP1/2) plays an essential role in the regulation of insulin signaling. Despite its high sequence homology with the other PDZ domains, only the PDZ1 domain of PTP-Bas showed distinct binding specificity for TAPP1/2. Although the interaction between PTP-Bas PDZ1 and TAPP1/2 is a therapeutic target for diabetes, the structural basis for the interaction has not been elucidated. In the present study, we determined the crystal structure of the PTP-Bas PDZ1 domain at 1.6 Å resolution. In addition, we calculated the structural models of complexes of PTP-Bas PDZ1 and the C-terminal peptides of TAPP1/2 (referred to as TAPP1p/2p). Structural comparison with the PTP-Bas PDZ2/RA-GEF2 peptide complex revealed a structural basis for distinct binding specificity of PTP-Bas PDZ1 for TAPP1p/2p peptides. Our high-resolution crystal structure of PTP-Bas PDZ1 will serve as a useful template for rational structure-based design of novel anti-diabetes therapeutics.

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene.

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT.

Expression of each cistron in the gal operon can be regulated by transcription termination and generation of a galk-specific mRNA, mK2.

The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378: 318-327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3' ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5' ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5' ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3' ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5' portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity.

The CnuK9E H-NS complex antagonizes DNA binding of DicA and leads to temperature-dependent filamentous growth in E. coli.

Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.

PI3K-ERK1/2 activation contributes to extracellular H2O2 generation in amyloid ß toxicity.

Amyloid ß peptide (Aß) induces hydrogen peroxide (H2O2) and superoxide generation, leading to neuronal death. Many studies have shown the involvement of NADPH oxidase, but the isotype-specific role was not assessed. Moreover, the activation status of phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 is unclear in extracellular H2O2 generation. In this paper, we showed that Aß1-42 induced extracellular H2O2 generation and the resulting cytotoxicity in a concentration-dependent manner. Nox2- and Nox4-specific siRNAs suppressed H2O2 and superoxide generation. LY294002 and U0126, inhibitors of PI3K and ERK1/2, respectively, reduced H2O2 generation in concentration-dependent manners. Furthermore, PI3K activation is responsible for ERK1/2 phosphorylation. An additional increase in H2O2 generation and corresponding cytotoxicity was observed after treatment with Aß1-42 and glutamate. These results suggest that Aß1-42 enhances the neuronal vulnerability to oxidative injury in Alzheimer's disease (AD) by increasing H2O2 generation.

XAGE-1a and XAGE-1d are potential biomarkers of lung squamous cell carcinoma.

BACKGROUND: Lung cancer is the leading cause of cancer deaths worldwide. We evaluated the diagnostic potential of sera XAGE-1a and XAGE-1d in lung cancer, both of which are variants of the X antigen family, member 1. METHODS: The expression levels of XAGE-1a and XAGE-1d in cell lines were determined using western blot analysis. Competitive ELISA was used to analyze XAGE-1a and XAGE-1d levels in culture supernatants and sera from 194 lung cancer patients and 194 healthy sex- and age-group-matched controls. To evaluate the diagnostic performance of these proteins, we also analyzed carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21-1) in culture supernatants and 388 sera using commercial ELISA kits. RESULTS: XAGE-1a and XAGE-1d proteins were expressed in both breast cancer and lung cancer cell lines, but they were only secreted by the latter. The areas under the curves (AUCs) for XAGE-1a and XAGE-1d were 0.787 and 0.806, respectively. The cutoff values (sensitivity, specificity) for XAGE-1a and XAGE-1d were 1.62 ng/ml (0.866, 0.572) and 2.51 ng/ml (0.871, 0.613), respectively. The diagnostic performance was improved for patients with squamous cell carcinoma. The AUC values for XAGE-1a and XAGE-1d for patients with squamous cell carcinoma versus a group containing all healthy participants and patients with any illness other than squamous cell carcinoma were similar to those for CEA and CYFRA 21-1. Better performance (AUC: 0.914) for all patients was obtained when using a combination of four markers (Random Forest). CONCLUSIONS: Sera XAGE-1a and XAGE-1d are potential biomarkers for lung cancer; they display a diagnostic performance comparable to that of CEA or CYFRA 21-1. Further studies are needed to evaluate the diagnostic and prognostic potential of XAGE-1a and XAGE-1d in lung cancer.

A mutational study of Cnu reveals attractive forces between Cnu and H-NS.

Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the mechanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.

In vivo transcription dynamics of the galactose operon: a study on the promoter transition from P1 to P2 at onset of stationary phase.

Quantitative analyses of the 5' end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD(600) 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD(600) 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population.