記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.biochi.2014.11.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3' end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3' end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.

DOI： 10.1093/nar/gku1069

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media SA  

DOI： 10.3389/fmicb.2014.00156

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

The ribosomes stalled at the end of non-stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non-productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans-translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome-tethered peptidyl-tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl-tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl-tRNAs located in the P-site of the stalled ribosome. This reaction required the GGQ (GlyGlyGln) hydrolysis motif, but not the SPF (SerProPhe) codonrecognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A-site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl-tRNA in a GGQ motif-dependent but codon-independent manner. In support of this model, the ArfA-RF2 pathway did not act on the SecM-arrested ribosome, which contains an aminoacyl-tRNA in the A-site.

DOI： 10.1111/j.1365-2958.2012.08190.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a "nascentome.'' We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.

DOI： 10.1371/journal.pone.0028413

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掲載種別：研究論文（学術雑誌）  

There are three classes of promoters for flagellar operons in Salmonella. Class 2 promoters are transcribed by σ 70 RNA polymerase in the presence of an essential activator, FlhD 4C 2, and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ 28 RNA polymerase and repressed by an anti-σ 28 factor, FlgM. σ 28 (FliA) and FliZ are encoded by the fliA and fliZ genes, respectively, which together constitute an operon transcribed in this order. This operon is transcribed from both class 2 and class 3 promoters, suggesting that it should be activated by its own product, σ 28, even in the absence of FlhD 4C 2. However, σ 28-dependent transcription occurs in vivo only in the presence of FlhD 4C 2, indicating that transcription from the class 2 promoter is a prerequisite to that from the class 3 promoter. In this study, we examined the effects of variously modified versions of the fliA regulatory region on transcription and translation of the fliA gene. We showed that FliA is not significantly translated from the class 3 transcript. In contrast, the 5'-terminal AU-rich sequence found in the class 2 transcript confers efficient fliA translation. Replacement of the Shine-Dalgarno sequence of the fliA gene with a better one improved fliA translation from the class 3 transcript. These results suggest that the 5'-terminal AU-rich sequence of the class 2 transcript may assist ribosome binding. FliZ was shown to be expressed from both the class 2 and class 3 transcripts. © 2011, American Society for Microbiology.

DOI： 10.1128/JB.05909-11

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GENETICS SOC JAPAN  

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

DOI： 10.1266/ggs.86.151

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

P>Accumulation of stalled ribosomes at the 3' end of mRNA without a stop codon (non-stop mRNA) is supposed to be toxic to bacterial cells. Escherichia coli has at least two distinct systems to rescue such stalled ribosomes: SsrA-dependent trans-translation and ArfA-dependent ribosome rescue. Combination of the ssrA and arfA mutations is synthetically lethal, suggesting the significance of ribosome rescue. In this study, we identified the E. coli yaeJ gene, encoding a peptide-release factor homologue with GGQ motif, as a multicopy suppressor of the lethal phenotype of ssrA arfA double mutant. The YaeJ protein was shown to bind to ribosomes. Both in vivo and in vitro, YaeJ showed the ribosome-rescue activity and promoted the hydrolysis of peptidyl-tRNA residing in the stalled ribosome. Missense mutation in the GGQ motif or deletion of the C-terminal unstructured tail abolished both the suppressor activity for ssrA arfA synthetic lethality and the ribosome-rescue activity, suggesting the importance of these structural features. On the basis of these observations, we propose that YaeJ acts as a stop codon-independent peptidyl-tRNA hydrolysing factor through binding to ribosomes stalled at the 3' end of non-stop mRNAs. It was also suggested that ArfA and YaeJ rescue the stalled ribosomes by distinct mechanisms.

DOI： 10.1111/j.1365-2958.2011.07607.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(4)C(2) heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium. This nutritional control was shown to be executed at a step after class 1 transcription. We isolated five Tn5 insertion mutants in which the class 2 expression was derepressed in poor medium. These insertions were located in the ydiV (cdgR) gene or a gene just upstream of ydiV. The ydiV gene is known to encode an EAL domain protein and to act as a negative regulator of flagellar expression. Gene disruption and complementation analyses revealed that the ydiV gene is responsible for nutritional control. Expression analysis of the ydiV gene showed that its translation, but not transcription, was enhanced by growth in poor medium. The ydiV mutation did not have a significant effect on either the steady-state level of flhDC mRNA or that of FlhC protein. Purified YdiV protein was shown in vitro to bind to FlhD(4)C(2) through interaction with FlhD subunit and to inhibit its binding to the class 2 promoter, resulting in inhibition of FlhD(4)C(2)-dependent transcription. Taking these data together, we conclude that YdiV is a novel anti-FlhD(4)C(2) factor responsible for nutritional control of the flagellar regulon.

DOI： 10.1128/JB.01494-10

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

P>Although SsrA(tmRNA)-mediated trans-translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in-frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome-rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA-arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis-refractory tag through trans-translation, indicating that ArfA-deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the Delta arfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon-independent mimic of aminoacyl-tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non-stop mRNA, which was otherwise SsrA-tagged and degraded. In vitro, purified ArfA acted on a ribosome-nascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3' end of a non-stop mRNA without involving trans-translation.

DOI： 10.1111/j.1365-2958.2010.07375.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GENETICS SOC JAPAN  

SsrA is a small RNA playing a crucial role in trans-translation, which leads to rescue of stalled ribosomes on or at the end of mRNA and addition of the degradation tag to a growing polypeptide. The lack of SsrA has been shown to enhance the temperature-sensitive (ts) phenotype of an E. coli strain defective in the degP gene, which encodes one of the periplasmic proteases. This severe ts phenotype was relieved only partially by an SsrA Do variant, which can lead to ribosome rescue but adds a protease-resistant tag instead of the degradation tag, suggesting that accumulation of polypeptides programmed by truncated mRNAs is responsible for growth defect of the ssrA degP mutant. Expression of an S210A-mutant DegP protein, which lacks the protease activity but retains the chaperone activity, could relieve the ts phenotype of the double mutant, suggesting that the chaperone activity but not the protease activity of DegP is required for growth of the ssrA-deficient cells at high temperature. Overexpression of the rpoE gene, which encodes sigma(E) responsible for the expression of factors involved in extracellular stress response, also suppressed the ts phenotype of the ssrA degP mutant. This suggests that the stress-responsing pathway(s) may be involved in the enhancement of ts phenotype of degP mutant in the absence of SsrA.

DOI： 10.1266/ggs.84.15

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes,fliC and flijB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation,fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.

DOI： 10.1128/JB.188.3.950-957.2006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CAMBRIDGE UNIV PRESS  

Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated Lacl tagging (Abo et al., EMBO J, 2000 19:3762-3769), we found that a variant Lacl (LaclDeltaC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of LaclDeltaC1 occurred efficiently without Lacl binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of LaclDeltaC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.

DOI： 10.1017/S1355838202020198

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING LTD  

Background: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging.
Results: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3' end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells.
Conclusion: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.

DOI： 10.1046/j.1365-2443.2002.00549.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING LTD  

Backgrounds: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C-terminus of the nascent polypeptide when a ribosome is stalled at the 3' end of an mRNA lacking a stop codon. This process, called trans-translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP-dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans-translation in Escherichia coli.
Results: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Pho-independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3' end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA.
Conclusion: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.

DOI： 10.1046/j.1365-2443.2002.00537.x

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DOI： 10.1093/emboj/19.14.3762

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

The trinucleotide repeats (CAG)(n) and (GGG)(n) have been shown to be expanded in responsible genes of several human hereditary neurological disorders. In studies of mice, we previously identified two homologous single-stranded (ss)(CAG) and ss(CGG) repeat-binding proteins, CAGER-1 (44 kDa) and CAGER-2 (40 kDa) (CAG-element-recognizing proteins). The specific binding activities of these proteins were predominantly detected in the mouse brain. We have isolated the cDNAs encoding CAGER-1 and CAGER-2 and found that they were identical to previously reported cDNAs for Pur alpha and Pur beta, respectively. Pur alpha of 28 kDa was previously identified as a replication-origin-binding protein that is ubiquitously expressed in proliferating cells. We show that the transcripts of CAGERs increase after birth and are detected at high levels in the adult mouse brain but at very low or virtually undetectable levels in other mouse tissues. Biochemical properties and molecular weights are different between CAGERs and Pur alpha/beta. Immunostaining with specific antibodies against CAGERs indicates that CAGERs in the mouse brain reside in nonproliferating neurons but not in proliferating glia. We conclude that CAGERs and Pur alpha/beta are unrelated proteins, and CAGERs are neuronal single-stranded sequence-binding proteins in the mouse brain. Misassignment of cDNAs is described. (C) 1999 Academic Press.

DOI： 10.1006/excr.1999.4603

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Background: Rho-independent terminators in Escherichia coli are DNA sequences of 30-50 bp consisting of a GC-rich dyad symmetry sequence followed by a run of T residues in the nontemplate strand. The transcription termination at the Rho-independent terminator occurs within the T-tract in vitro. It has been believed that the transcription termination at the Rho-independent terminator occurs within the T-tract in vivo, as established in vitro, and therefore the 3' ends of mRNAs are mostly generated as a direct result of transcription termination. However, how the transcription termination occurs and how the 3' ends of mRNAs are formed in living cells remains to be studied.
Results: We developed a double terminator system in which a second Rho-independent terminator was placed downstream of the crp terminator. This system made it possible to detect transcripts that pass through the cry terminator by Northern blotting. We found that most of the crp transcripts extend beyond the cry terminator. The transcriptional read-through at the cry terminator was reduced when the translation of cry mRNA was interrupted. The level of the read-through transcript decreased with distance between the two terminators, suggesting that transcription termination occurs at multiple positions beyond the crp terminator.
Conclusion: We conclude that most RNA polymerase reads through the cry terminator in the natural situation and terminates transcription over a wide region downstream of the crp terminator, resulting in heterogeneous primary transcripts that are subsequently processed back to the terminator hairpin. We propose that ribosome translation to the cry stop codon causes read-through of the terminator. The regulatory effect of translation on Rho-independent termination may be a general phenomenon at other operons.

DOI： 10.1046/j.1365-2443.1999.00246.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE LTD  

Background: The plasmid R100 encodes tra genes essential for conjugal DNA transfer in Escherichia coli. Genetic evidence suggests that the traJ gene encodes a positive regulator for the traY-I operon, which includes almost all the tva genes located downstream of traJ. The molecular mechanism of regulation by TraJ, however, is not yet understood. traY is the most proximal gene in the traY-I operon. TraY promotes DNA transfer by binding to a site, sbyA, near the origin of transfer. TraY is suggested to have another role in regulation of the traY-I operon, since it binds to two other sites, named sbyB and sbyC, located in the region preceding traY-I.
Results: Using a traY-lacZ fusion gene, we showed that the traY-I operon was expressed only in the presence of traJ. The TraJ-dependent expression of traY-I required the E. coli arcA gene, which encodes a host factor required for conjugation. TraJ-dependent transcription occurred from a promoter (named pY) located upstream of traY-I. The isolated TraJ protein was found to bind to a dyad symmetry sequence, named sbj (specific binding site of TraJ), which existed in the intergenic region between traJ and traY-I. We also demonstrated that TraY repressed the TraJ-dependent expression of traY-I at the TraY binding sites, sbyB and sbyC, which overlapped with pY.
Conclusions: TraJ is a protein which binds to the sbj site in the region upstream of the promoter pY and positively regulates expression of the traY-I operon in the presence of the E. coli arcA gene. Since sbj is located 93 bp upstream of pY in the intergenic region between traJ and traY-I, TraJ presumably contacts with a transcription apparatus to promote transcription from pY. TraY, which is known to activate the initiation of conjugal DNA transfer, has a new role in the transcriptional autoregulation of traY-I expression. At levels which are sufficient to initiate conjugal DNA transfer, TraY represses traY-I transcription in the presence of TraJ.

DOI： 10.1046/j.1365-2443.1998.00194.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

We have previously identified three sites, named sbi, ihfA, and sbyA, specifically recognized or bound by the TraI, IHF, and TraY proteins, respectively; these sites are involved in nicking at the origin of transfer, oriT, of plasmid R100. In the region next to these sites, there exists the sbm region, which consists of four sites, sbmA, sbmB, sbmC, and sbmD; this region is specifically bound by the TraM protein, which is required for DNA transfer. Between sbmB and sbmC in this region, there exists another IHF-binding site, ihfB. The region containing all of these sites is located in the proximity of the tra region and is referred to as the oriT region. To determine whether these sites are important for DNA transfer in vivo, rye constructed plasmids with various mutations in the oriT region and tested their mobilization in the presence of R100-1, a transfer-proficient mutant of R100. Plasmids with either deletions in the sbi-ihfA-sbyA region or substitution mutations introduced into each specific site in this region were mobilized at a greatly reduced frequency, showing that all of these sites are essential for DNA transfer. By binding to ihfA, IHF, which is known to bend DNA, may be involved in the formation of a complex (which may be failed oriT-some) consisting of TraI, IHF, and TraY that efficiently introduces a nick at oriT. Plasmids with either deletions in the sbm-ihfB region or substitution mutations introduced into each specific site in this region were mobilized at a reduced frequency, showing that this region is also important for DNA transfer. By binding to ihfB, IHF may also be involved in the formation of another complex (which may be called the TraM-IHF complex) consisting of TraM and IHF that ensures DNA transfer with a high level of efficiency. Several-base-pair insertions into the positions between sbyA and sbmA affected the frequency of transfer in a manner dependent upon the number of base pairs, indicating that the phasing between sbyA and sbmA is important. This in turn suggests that both OriT-some and the TraM-IHF complex should be in an appropriate position spatially to facilitate DNA transfer.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, TraI and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the TraI protein and Mg2+. The products were complex DNA molecules with a protein covalently linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the TraI protein alone, indicating that the protein attached at the 5' end of the nick is the TraI protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA indicates that DNA bending is important in the formation of the complex including the TraI and TraY proteins at oriT.

DOI： 10.1093/oxfordjournals.jbchem.a124604

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated p(M1) and p(M2). Promoter p(M2) lies upstream of p(M1) and overlaps the sbmC-sbmD region. Promoter p(M1) is constitutively expressed, while p(M2) is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near p(M2). The traM gene is likely to be expressed from p(M2) when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from p(M2) Could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.

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記述言語：英語   出版者・発行元：MICROBIOL RES FOUNDATION  

DOI： 10.2323/jgam.36.287

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：GENETICS SOC JAPAN  

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記述言語：日本語  

CiNii Article

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開催年月日： 2010年9月20日 - 2010年9月22日

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