A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

A conformational unfolding reaction activates phage fd for the infection of Escherichia coli

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

Structural model of the R state of Escherichia coli aspartate transcarbamoylase with substrates bound

The allosteric enzyme aspartate transcarbamoylase (ATCase) exists in two conformational states. The enzyme, in the absence of substrates is primarily in the low-activity T state, is converted to the high-activity R state upon substrate binding, and remains in the R state until substrates are exhausted. These conformational changes have made it difficult to obtain structural data on R-state active-site complexes. Here we report the R-state structure of ATCase with the substrate Asp and the substrate analog phosphonoactamide (PAM) bound. This R-state structure represents the stage in the catalytic mechanism immediately before the formation of the covalent bond between the nitrogen of the amino group of Asp and the carbonyl carbon of carbamoyl phosphate. The binding mode of the PAM is similar to the binding mode of the phosphonate moiety of N-(phosphonoacetyl)-l-aspartate (PALA), the carboxylates of Asp interact with the same residues that interact with the carboxylates of PALA, although the position and orientations are shifted. The amino group of Asp is 2.9 A away from the carbonyl oxygen of PAM, positioned correctly for the nucleophilic attack. Arg105 and Leu267 in the catalytic chain interact with PAM and Asp and help to position the substrates correctly for catalysis. This structure fills a key gap in the structural determination of each of the steps in the catalytic cycle. By combining these data with previously determined structures we can now visualize the allosteric transition through detailed atomic motions that underlie the molecular mechanism.     

Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.     

Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain

Glutamate 5-kinase (G5K) catalyzes the controlling first step of the synthesis of the osmoprotective amino acid proline, which feed-back inhibits G5K. Microbial G5K generally consists of one amino acid kinase (AAK) and one PUA (named after pseudo uridine synthases and archaeosine-specific transglycosylases) domain. To investigate the role of the PUA domain, we have deleted it from Escherichia coli G5K. We show that wild-type G5K requires free Mg for activity, it is tetrameric, and it aggregates to higher forms in a proline-dependent way. G5K lacking the PUA domain remains tetrameric, active, and proline-inhibitable, but the Mg requirement and the proline-triggered aggregation are greatly diminished and abolished, respectively, and more proline is needed for inhibition. We propose that the PUA domain modulates the function of the AAK domain, opening the way to potential PUA domain-mediated regulation of G5K; and that this domain moves, exposing new surfaces upon proline binding.     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orf7和orf5基因片段。利用EcoRI、SpeI和HindIIl位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMDl8NE,并比较所克隆基因序列的同源性。将串联的orf7和orf5基因亚克隆到原核表达载体pGEX．KG,构建重组原核融合表达质粒pGEX．KGNE,并转化BL21,SDS．PAGE和Western．blot分析表明：orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础。     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orr7和orf5基因片段.利用EcoRI、SpeⅠ和Hind Ⅲ位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMD18NE,并比较所克隆基因序列的同源性.将串联的orf7和orf5基因亚克隆到原核表达载体pGEX-KG,构建重组原核融合表达质粒pGEX-KGNE,并转化BL21,SDS-PAGE和Western-blot分析表明orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础.     

Widespread occurrence of power-law distributions in inter-repeat distances shaped by genome dynamics

Repetitive DNA sequences derived from transposable elements (TE) are distributed in a non-random way, co-clustering with other classes of repeat elements, genes and other genomic components. In a previous work we reported power-law-like size distributions (linearity in log-log scale) in the spatial arrangement of Alu and LINE1 elements in the human genome. Here we investigate the large-scale features of the spatial arrangement of all principal classes of TEs in 14 genomes from phylogenetically distant organisms by studying the size distribution of inter-repeat distances. Power-law-like size distributions are found to be widespread, extending up to several orders of magnitude. In order to understand the emergence of this distributional pattern, we introduce an evolutionary scenario, which includes (i) Insertions of DNA segments (e.g., more recent repeats) into the considered sequence and (ii) Eliminations of members of the studied TE family. In the proposed model we also incorporate the potential for transposition events (characteristic of the DNA transposons' life-cycle) and segmental duplications. Simulations reproduce the main features of the observed size distributions. Furthermore, we investigate the effects of various genomic features on the presence and extent of power-law size distributions including TE class and age, mode of parental TE transmission, GC content, deletion and recombination rates in the studied genomic region, etc. Our observations corroborate the hypothesis that insertions of genomic material and eliminations of repeats are at the basis of power-laws in inter-repeat distances. The existence of these power-laws could facilitate the formation of the recently proposed "fractal globule" for the confined chromatin organization.     

The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.     

Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F' partial diploid Escherichia coli.

The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F' partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His-) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombination were determined by measuring the frequency of His- homogenotes, eg. F' hisC780, hisI+/hisC780, hisI+, arising from a His+ heterogenote, F' hisC780 hisI+/hisC+, his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F' plasmid (exogenote) of the parental hetergenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase the frequency of nonreciprocal recombination. A defect in the ability to excise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. Mutagenic agents, with the exception of ethyl methanesulfonate (EMS), induced greater increases in recombination than the chemotherapeutic agents or the polA1 mutation. EMS, which causes relatively little degradation of DNA, was more mutagenic but less recombinogenic than MMS, a homologous compound ths that inhibition of DNA occurring single-stranded regions in replicative intermediates of the DNA. Mutagens which cause the rapid breakdown of DNA may, in addition, introduce lesions into the genome that increase the number of single-stranded regions thus inducing even higher frequencies of recombination.     

Immunoelectron microscopy of ribosomes carrying a fluorescence label in a defined position. Location of proteins S17 and L6 in the ribosome of Escherichia coli

By coupling fluorescein to a defined amino acid of a single ribosomal protein and incorporating this protein into the ribosome, we have obtained ribosomes labelled at a single, defined position. A fluorescein-specific antibody preparation was used to locate the fluorescein residues bound to the two cysteines at positions 58 and 63 of protein S17 and to the cysteine at position 86 of protein L6. This study demonstrates the advantages which accrue from the combination of electron microscopy and fluorimetry.     

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn²⁺. Inhibition of liposome-bound bovine cytochrome oxidase by external Zn²⁺ titrates with a K_i of 1 ± 0.3 μM. Presumably, the Zn²⁺-binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of Cu_B is tentatively proposed to be responsible for the effect.