DNA structural variations in the E. coli tyrT promoter

X-ray studies have established that the structure of a right-handed, Watson-Crick double helix can change from place to place along its length as a function of base sequence. The base pairs transmit deformations out to the phosphate backbone, where they can then be recognized by proteins and other DNA-binding reagents. Here we have examined at single-bond resolution the interactions of three commonly used nucleases (DNAase I, DNAase II, and copper-phenanthroline) with a DNA of natural origin, the 160 bp tyrT promoter. All three of these reagents seem sensitive to DNA backbone geometry rather than base sequence per se. Their sequence-dependent patterns of cleavage provide evidence for structural polymorphism of several sorts: global variation in helix groove width, global variation in radial asymmetry, and local variation in phosphate accessibility. These findings explain how sequence zones of a certain base composition, or purine-pyrimidine asymmetry, can influence the recognition of DNA by protein molecules.     

tRNA-binding centers of Escherichia coli ribosomes and their structural organization

Problems concerning the interaction of tRNA with Escherichia coli ribosomes in different functional states were studied. These problems deal first of all with the number of tRNA-binding sites on ribosome, the conservation of the codon-anticodon interaction at the P-site and with regions of tRNA interacting with ribosome. The problems concerning structural organization of tRNA-binding centers are discussed in more detail.     

Compilation and analysis of Escherichia coli promoter DNA sequences.

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.     

DNA methylation influences trpR promoter activity in Escherichia coli K-12

Summary Methylation of adenine in the GATC-sequence of the-35 region of the trpR promoter decreases activity by 2–3 fold.     

Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene

The constitutive low-efficiency promoter site (P₂) near the middle of the tryptophan operon of Escherichia coli has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internal promoter with those which retain it show that P₂ is located within trpD, the region coding for phosphoribosyl anthranilate transferase. P₂ maps near the operator-distal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P₂ site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trp A polypeptides present in repressed cells.     

Flexibility of the DNA enhances promoter affinity of Escherichia coli RNA polymerase.

Two types of mechanisms are discussed for the formation of active protein-DNA complexes: contacts with specific bases and interaction via specific DNA structures within the cognate DNA. We have studied the effect of a single nucleoside deletion on the interaction of Escherichia coli RNA polymerase with a strong promoter. This study reveals three patterns of interaction which can be attributed to different sites of the promoter, (i) direct base contact with the template strand in the '-35 region' (the 'recognition domain'), (ii) a DNA structure dependent interaction in the '-10 region' (the 'melting domain'), and (iii) an interaction which is based on a defined spatial relationship between the two domains of a promoter, namely the 'recognition domain' and the 'melting domain'.     

Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions in recombinant plasmids in Escherichia coli

The ability to clone a variety of sequences with varying capabilities of adopting non-B structures (left-handed Z-DNA, cruciforms or triplexes) into three loci of pBR322 was investigated. In general, the inserts were stable (non-deleted) in the EcoRI site (an untranslated region) of pBR322. However, sequences most likely to adopt left-handed Z-DNA or triplexes in vivo suffered deletions when cloned into the BamHI site, which is located in the tetracycline resistance structural gene (tet). Conversely, when the promoter for the tet gene was altered by filling-in the unique HindIII or ClaI sites, the inserts in the BamHI site were not deleted. Concomitantly, the negative linking differences of the plasmids were reduced. Also, inserts with a high potential to adopt Z-DNA conformations were substantially deleted in the PvuII site of pBR322 (near the replication origin and the copy number control region), but were less deleted if the tet promoter was insertion-mutated. The deletion phenomena are due to the capacity of these sequences to adopt left-handed Z-DNA or triplexes in vivo since shorter inserts, less prone to form non-B DNA structures, or random sequences, did not exhibit this behavior. Sequences with the potential to adopt cruciforms were stable in all sites under all conditions. These results reveal a complex interrelationship between insert deletions (apparently the result of genetic recombination), negative supercoiling, and the formation of non-B DNA structures in living Escherichia coli cells.     

Macrodomain organization of the Escherichia coli chromosome

We have explored the Escherichia coli chromosome architecture by genetic dissection, using a site-specific recombination system that reveals the spatial proximity of distant DNA sites and records interactions. By analysing the percentages of recombination between pairs of sites scattered over the chromosome, we observed that DNA interactions were restricted to within subregions of the chromosome. The results indicated an organization into a ring composed of four macrodomains and two less-structured regions. Two of the macrodomains defined by recombination efficiency are similar to the Ter and Ori macrodomains observed by FISH. Two newly characterized macrodomains flank the Ter macrodomain and two less-structured regions flank the Ori macrodomain. Also the interactions between sister chromatids are rare, suggesting that chromosome segregation quickly follows replication. These results reveal structural features that may be important for chromosome dynamics during the cell cycle.     

Effect of DNA structural flexibility on promoter strength--molecular dynamics studies of E. coli promoter sequences

To study possible correlations between promoter activity and the structural flexibility of the DNA helix, we have carried out unrestrained molecular dynamics simulations of the -10 consensus region sequence and five variants forming the -10 region of various Escherichia coli promoter sequences. Analyses of the trajectories obtained from the simulations show that the consensus sequence has a pattern of two structurally flexible nucleotide steps sandwiched between two stiff steps. In the other sequences, this pattern varies in consonance with the change in the sequence. The variations in the patterns show correlation with the promoter strength.     

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.     

Levels of glutathione in Escherichia coli.

Log phase cells of Escherichia coli growing in minimal medium contain a basal level of glutathione (5 pmol/mL per Klett unit) which can increase more than sixfold when the cells reach stationary phase. Since the addition of cysteine alone to log phase cells illicits the same response, the increase in the intracellular pool of glutathione appears to be influenced by the amount of cysteine available for glutathione synthesis. Glucose depletion at low cell densities resulted in a decrease in the glutathione pool while the addition of amino acids other than cysteine did not affect the glutathione pool. Depletion of ammonia or proline as the nitrogen source also resulted in a decrease in the glutathione pool to one-third of the original basal levels as did a shift to anaerobic growth. The large glutathione pool in stationary phase cells dropped from 31.5 to 4.5 pmol/mL per Klett unit within 30 min of transfer to fresh medium. There was no apparent correlation between changes in the glutathione and coenzyme A--glutathione disulfide (CoASSG) pools after a variety of metabolic disruptions.     

Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator

A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites. Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids. The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests. The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence. Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site. These derivatives showed in vivo operator activity. Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed. These plasmids should be useful in preparing large amounts of the 41-bp fragment.