摘要:

SummaryEscherichia coliRho factor Is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine‐containing RNA target sites, subsequent RNA‐dependent ATP hydrolysis, and an RNA‐DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination functionin vitroandin vivohave been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revela

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00376.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryTheBacillus subtilistryptophan (trpEDCFBA) operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L‐tryptophan, a transcription terminator forms and the operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the operon. The mechanism of selection between these alternative structures involves atrans‐acting RNA‐binding regulatory protein. This protein is the product of themtrBgene and is called TRAP for trp attenuation p/rotein. TRAP has been shown to bind specifically totrpleader RNA, and to cause transcription of thetrpoperon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is f

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00377.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryRecent footprinting, sedimentation and neutron‐scattering results obtainedIn vivoor on pre‐translocation and post‐translocation ribosomal complexes are integrated with cross‐linking and immunoelectron microscopy information, it is proposed that the 30S subunit pulses during translocation and that its preand post‐translocation structures are not necessarily identical. Accordingly, transiocation is characterized by three consecutive conformational states of the 30S and 50S subunits. State 1 (the pre‐translocation state) lasts until the elongation factor EF‐G·GTP complex binds to the ribosome or adopts the GTPase conformation. State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF‐G adopts a subsequent conformation or is released from the ribosome. State 3 (the post‐translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl‐tRNA. In state 2, 16S RNA hairpins 26 and 33‐33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed. A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl‐tRNA elbow from the A to the P site, around a (retractable?) S19 domain. These twists result in an unlocking of the platform and the head from the 50S subunit. Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem‐loops. These twisted or otherwise‐exposed residues readopt their previous setting upon completion of translocation, i.e. states 1 and 3 of 16S RNA differ more from state 2 than from each other. Yet the ribosome is never fully locked or unlocked at any time during elongation. It is unlocked in one or another respect in the pre‐ and post‐translocation states, and unlocked to the largest extent in state 2.‘Le plus grand Phénomène de la Nature, le plus Merveilleux, est le Mouvement’ (The greatest, the most wonderful Phenomenon in Nature

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00378.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe formation of cyclopropane fatty acids (CFAs) inEscherichia coliis a post‐synthetic modification of the phospholipid bilayer that occurs predominantly as cultures enter the stationary phase of growth. The mechanism of this growth phase‐dependent regulation of CFA synthesis was unclear, since log‐phase and stationary‐phase cultures had been reported to contain similar levels of the enzyme catalysing the reaction (CFA synthase). We report that the timing of CFA synthesis can be explained by two unusual features. First, the gene encoding CFA synthase (cfa) was found to be transcribed from two promoters and the 5′ ends of both transcripts were mapped by primer extension. One of the promoters was active only during the log‐to‐stationary phase transition and depended on the putative sigma factor encoded by therpoS(katF)gene whereas the other promoter had a standard σ70promoter consensus sequence and was expressed throughout the growth curve. Second, CFA synthase activity was shown to be unstablein vivoand a Cfa fusion protein was found to have a half life of<5min. The combination of these factors meant that, although CFA synthase was synthesized throughout the growth curve, a large increase in activity occurred during the log‐to‐stationary phase transition. As stationary phase progressed, the Increased CFA synthase activity rapidly declined to the basal level. This transient increase In CFA synthase activity coupled with the cessation of net phospholipid synthesis in stationary phase provides an explanation for the unusual time cours

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00379.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe activity ofEscherichia colipyruvate oxidase (PoxB) was shown to be growth‐phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functionalrpoS(katF)gene which encodes a σ factor required to transcribe a number of stationary‐phase‐induced genes. PoxB activity as well as the β‐galactosidase encoded by apoxB::lacZprotein fusion was completely abolished in a strain containing a defectiverpoSgene. Northern and primer extension analyses showed thatpoxBexpression was regulated at the transcriptional level and was transcribed from a single promoter; the 5′ end of the mRNA being located 27 bp upstream of the translational initiation codon ofpoxB.ThepoxBgene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genesarcA, arcBorfnrwere not involved in anaerobicpoxBgene expression. Expression of therpoS(katF)gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found thatpoxBnull mutations had no effect onrpoS(katF)expression. Inactivation of two genes involved In acetate metabolism,ackAandpta, had no effect on eitherpoxBorrpoS(katF

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00380.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryMaxicell labelling and two‐dimensional gel electro‐phoresis (2‐D PAGE) have identified the proteins encoded bysspAandsspB(SspA, SspB) as proteins D27.1 and A25.8, respectively, in theEscherichia coligene‐protein database.SspAexpression increases with decreasing growth rate and is induced by glucose, nitrogen, phosphate or amino acid starvation. The promoter, Pssp, is similar to gearbox promoters. Inactivation of SspA (sspA::neo) blockssspBexpression. [35S]‐methionine‐labelled proteins synthesized during growth and during stationary phase are different in δsspAstrains compared tosspAstrains. This difference is enhanced during extended stationary phase (24–72 h). Long‐term (10 d) viability of arginine‐starved isogenic strains shows thatsspAcultures remain viable significantly longer than δsspAmutants. 2‐D PAGE of proteins expressed during exponential growth shows that expression of at least 11 proteins is altered in δsspAstrains. A functionalrelAgene is required forsspAto af

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00381.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryBacteriophage T7 expresses a serine/threonine‐specific protein kinase activity during Infection of Its host,Escherichia coli.The protein kinase (gpO.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub‐optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphoryiated in T7‐infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high‐resolution two‐dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electro‐phoretic data moreover indicate that elongation factor P is phosphorylated in T7‐infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two‐dimensional gel pattern, and to confirm that serine is the phosphate‐accepting amino acid. The two‐dimensional gels show that thein vivoexpression of gp0.7 PK results in the phosphorylation of over 90 proteins, which Is a significantly higher number than previous estimates. The protein kinase activities of the T7‐related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00382.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryEscherichia coliinduces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide‐deficientphoAreporter sequence to acsi(carbonstarvation‐inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the clonedphoAfusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild‐type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locusslp(starvationlipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. Theslpgene was located at 78.6 centisomes on theE. coligenetic map. The ‐10 and ‐35 regions upstream of the mRNA start site were characteristic of a σ70promoter. The major transcript from this promoter was sufficiently large to containslpsequences but not the downstream open reading frame. Induction of β‐galactosidase activity from aslp::lacZtranslational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation‐inducible or stationary phase‐i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00383.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummarySequencing of N‐terminal and internal peptide fragments of the purified 17kDaBacillus subtilispeptldyl‐prolylcls‐transisomerase (PPlase) revealed sequence identity to conserved regions of a number of eukaryotic and prokaryotic cyclophilins. Using two oligonucleotide primers corresponding to the N‐terminus and a highly conserved internal amino acid sequence, polymerase chain reactions (PCR) withB. subtilisgenomic DNA were carried out. The resultant PCR fragment of 335 bp was cloned, sequenced and subsequently used as a probe for screening a λZap II gene library of B. subtilis. Two overlapping positive clones of 5 and 7 kb containing theB. subtilisPPlase gene (ppiB), which is 432 bp in length and encodes a protein of 144 amino acid residues, were identified and two distinct transcriptional initiation sites at the 5′ end ofppiBwere mapped. The entire region (35 kb) betweenspoVAandserAwas recently sequenced inB. subtilis, and an open reading frame (ORF) that encodes a putative peptidyl‐prolylcis‐transisomerase at about 210° on theB. subtilisgenetic map was located. This putative PPlase is identical to PPiB. We have overexpressed theppiBgene inEscherichia coli, purified the encoded protein to apparent homology and shown that it exhibits PPlase activity. In addition, the recombinant PPiB shows a significant inhibition of PPlase activity by cyclosporin A (CsA) at a level comparable to that observed for theB. subtilisenzyme. Interestingly theB. subtilisPPlase shows about 40% identity to eukaryotic PPlases and less similarity to those of Gram‐negative bacteria (27–32% identity). Like other interruption mutants of yeast and Neurospora, which iack a functionai cyclophilin gene, a B. subtiiis mutant containing ppiBv.cat, a caf‐interrupted copy of ppiB in the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00384.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe 18 kDa histone H1‐like protein fromChlamydia trachomatis(Hc1) is a DNA–binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 inEscherichia coliresults in an overall relaxation of DNA and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single‐stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant concentrations. These results were found to coincide with the formation of condensed Hc1–DNA and Hc1–RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1in vivoare

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1994.tb00385.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY