摘要:

SummaryTheEcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+: but in binding to DNA in the absence of Mg2+, theEcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+toEcoRV‐DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of theEcoRV endonuclease, and of its complexes with either 8pecific or non‐specific DNA, have been solved by X‐ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non‐specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B‐like structure. Since the free energy changes for the formation of specific and non‐specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, theEcoRV–DNA complex would be unable to bind Mg2+and thus unable to cleave DNA. The specificity of theEcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to whic

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01685.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryTranslation of the IS 10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS 10 mRNA using one particular IS 10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half‐life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half‐life. At wild‐type levels of translation initiation, the rate‐limiting step in physical decay of full length IS 10 message for a particular IS 10 derivative is shown to berne‐dependent endonucleolytic cleavage; 3′ exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations andrnefunction, together with the above observations, suggests that translation stabilizes messages in a general way againstrne‐dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in anrnemutant and involve the 3′ end of the transcript; we propose that these additional effects reflect translation‐mediated stimulation of t

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01686.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryA number of bacterial DNA‐binding proteins, including IS element transposases, act preferentially incis.We show below that the degree of preferentialcisaction by IS 10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation.Cispreference is increased several fold by mutations that decrease translation initiation, by the presence of IS 10‐specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely,cispreference is decreased by mutations that increase translation initiation; in some cases,cispreference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest thatcispreference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half‐life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01687.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryBacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII‐dependent PIpromoter is responsible for λintgene expression. The only apparent counterpart to PIin P22 is oriented in the opposite direction, and cannot transcribe the P22intgene. We show that this promoter, calledPal, is active bothin vivoandin vitro, and is dependent upon the P22 cII‐like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 pLpromoter and theintgene. The newly determined sequence is densely packed with genes from the pLdirection, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (pal) direction, and no additional binding motifs for the P22 c1 protein. We conclude thatintgene expression in P22 is regulated by a different mechanism tha

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01688.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryChlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non‐replicative elementary bodies (EBs) and replicatlve reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure inEscherichia coli.High‐level Hc1 expression inE. coliis self‐limiting and down‐regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub‐structural levels may have specific regulatory functions through its interaction with chromosomal DNA, InE. colithis is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis oflacZfusions with promoter regions sensitive to supercoiling suggests that low‐level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasm id DNA from bothE. coliandChlamydia trachomatissupports a decrease in superhelicity preceding nucleoid formation.In vitroanalysis of purified Hc1–DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1‐mediated changes in gene expression may precede metab

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01689.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryEscherichia coliRNA polymerase is composed of four different subunits, 2α, β, β′ and σ;. Among these subunits, the role of β′ is poorly understood. TherpoC10 mutation affecting β′ has been isolated as a suppressor mutation of the temperature‐sensitivenusA11 mutant. DNA sequence analysis revealed that therpoC10 mutant is a substitution of Lys for Glu‐402. This increased positive charge appears to compensate for the increased negative charge present in thenusA11 protein (Asp for Gly‐181).In vivomeasurements of reporter gene expression have revealed thatrpoC10 restores ρ‐dependent termination but fails to restore ρ‐independent termination innusA.11 Moreover, therpoC10 mutation, in combination with anynusAmutation, inhibited λ Q‐mediated antitermination without affecting N antitermination and severely restricted λ phage development. The inhibition of Q function and λ growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase β′ subunit plays a crucial rote in factor‐dependent transcriptio

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01690.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryThe signal sequence of theKlebsiella oxytoca pulGgene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me‐Phe) pilins. The significance of this similarity is confirmed by the observation that thepulOgene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino‐terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino‐terminal phenylaianine residue. Site‐directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino‐terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants ofPseudomonas aeruginosatype IV prepilin, which were processed but only inefficiently IV‐methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car‐boxy‐terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01691.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryTheYersinia enterocoliticaO:3 lipopotysaccharide O‐antigen is a homopotymer of 6‐deoxy‐L‐altrose. The clonedrfbregion was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementatton experiments showed that eight of the genes, organized into two operons,rfbABCandrfbDEFGH, are essential for 0‐antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar toSalmonellaproteins involved in the dTDP‐l‐rhamnose biosynthesis. Rhamnose and 6‐deoxy‐l‐altrose are C3‐epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT ofEscherichia coli.This and transposon mutagenesis showed that RfbD and RfbE function a

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01692.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryUrease genes fromHelicobacter feliswere cloned and expressed inEscherichia colicells. A genomic bank ofSau3A‐digestedH. felischromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen‐limiting medium. Subcloning of DNA from an urease‐positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min‐1mg‐1bacterial protein toE. coliHB101 bacteria grown on a nitrogen‐limiting medium. Random mutagenesis using a MiniTn3‐Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease‐positive phenotype inE. coiiclones. To localize the putative structural genes ofH. felison plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti‐H.felisrabbit serum. One mutant cione did not synthesize the putative UreB subunit ofH. felisurease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designatedureAandureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products ofHelico

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01693.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

SummaryA proportion (up to 20%) of newly arising streptomycin‐dependent (SmD) colonies ofEscherichia coliWP2 contain bacteria where, in addition to a known SmD‐determining (primary) mutation in therpsLgene, there is a further ancillary mutation in the same gene. These ancillary mutations occur at 10 sites between 11 and 201 bp away from the primary mutation. Ancillary mutations have been found in mutant colonies arising both spontaneously and after treatment with ultraviolet light and some have been found repeatedly. Ancillary mutations were frequently found to occur in mixed clones with an excess of bacteria carrying only the primary SmDmutation. No ancillary mutations were found in an adjacent non‐coding region and there were no coding sequence changes that did not alter the amino acid specified. Although a selective advantage for bacteria containing ancillary mutations could not be demonstrated directly in every case, some small advantage must be presumed to have occurred to explain the absence of mutations at the other sites and particularly at third (wobble) codon positions. Ancillary mutations appear to occur fairly early in the life of a newly arisen SmDmutant clone in some sort of hypermutable process. Whether they are noticed appears to depend on their conferment of some selective advantage on the bacteria carrying them. While the ancillary mutations withinrpsLlie close to, and may be consequent upon the formation of a primary SmDmutation, their mechanism of formation appears to be at least to some extent independent and does not involve therecAorumuCgenes. Ancillary mutations were also detected in bacteria lacking reverse transcriptase or transcription repair coupling f

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1993.tb01694.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY