Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator

The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.     

Interaction of the LexA repressor and the uvrC regulatory region

We have studied the in vitro interaction of the LexA repressor protein and the uvrC regulatory region. We find that there is specific binding to two regions, the region we have defined as lexA1 and the lexA2-lexA3 region. Our findings support the possibility of an inducible regulation for this complex operon.     

Model of a LexA repressor dimer bound to recA operator

A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.     

Improved model of a LexA repressor dimer bound to recA operator

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wild-type LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless.     

Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12.

The specificity of LexA protein binding was investigated by quantifying the repressibility of several mutant recA and lexA operator-promoter regions fused to the Escherichia coli galactokinase (galK) gene. The results of this analysis indicate that two sets of four nucleotides, one set at each end of the operator (terminal-nucleotide contacts), are most critical for repressor binding. In addition, our results suggest that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator have comparable effects on repressibility. The symmetry of this interaction justified reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus (5')CTGTATAT. Although the first four positions of this sequence were most important, the last four were well conserved among binding sites and appeared to modulate repressor affinity. The role of the terminal-nucleotide contacts and the mechanism by which the internal sequences affected repressor binding are discussed.     

Interaction of the trp repressor with trp operator DNA fragments

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor l-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpO ^c, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the ³¹P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.Abbreviations CD circular dichroism - trpO, trpR aroH trp operator fragments - trpO ^c trpMH mutant trp operator fragments     

The complete nucleotide sequence of the I-E alpha d immune response gene.

We have isolated and sequenced the complete murine I-E alpha immune response gene of the H-2db haplotype. The I-E alpha d gene consists of 5300 basepairs and is organized into five or possibly six exons that correspond to different domains of the alpha chain. The amino acid sequence deduced from the I-E alpha gene shows 75% homology to its human counterpart, the HLA-DR alpha chain. The absence of I-E antigen in H-2 mice is due to lack of E alpha chain synthesis. We show here that this defect is caused by a deletion in the 5' end of the I-E alpha b gene.     

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

The complete nucleotide sequence of a rice 25S.rRNA gene

The complete nucleotide (nt) sequence of a rice nuclear 25S.rRNA gene has been determined. The 25S.rRNA-coding region is 3377 bp long. The G + C content is 59.4%. The structural organization of this rRNA is very similar to that of yeast 26S rRNA.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

The complete nucleotide sequence of a rice 17S rRNA gene.

The complete nucleotide sequence of a rice nuclear 17S rRNA gene (rDNA) has been determined. The rice rDNA is 1812 bp long and its G + C content is 51.3%. This nucleotide sequence shows 79%, 80% and 80% homology to those of yeast, Xenopus laevis and rat 18S rDNAs, respectively. Divergency of nucleotide sequences is largely attributed to five blocks of highly variable regions, where eukaryotic specific sequences can be observed.     

Interaction of the bacteriophage P22 Arc repressor with operator DNA

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).