Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control.

CysB is a tetrameric LysR-type transcriptional regulator that acts as an activator of cys regulon genes and as an autorepressor. Positive control of cys genes requires the presence of the inducer N-acetylserine. Following random and site-directed mutagenesis of the cysB gene, 20 CysB variants were isolated. Six single amino acid substitutions within the N terminus of CysB abolished the DNA-binding ability of the protein. Seven mutations in the central region of CysB affected its response to the inducer. Four of these CysB mutants retained repressing activity, but lost their activating function in vivo. Their DNA binding characteristics were consistent with an inability to respond to acetylserine by a qualitative change in the DNA-protein interaction. Three of the single residue substitutions resulted in constitutive activity of CysB. The electrophoretic mobility of the complex formed by one of the CysBc variants with the cysP promoter suggested a dimeric state of this protein. Characteristics of six truncated CysB variants lacking 5-30 C-terminal residues indicated the involvement of the C terminus in the DNA binding, oligomerization, and stability of CysB. The single substitution Y27G resulted in the CysBpc variant, able to bind DNA and to respond to the inducer by a qualitative change in the DNA-protein complex, but defective in the positive control of the cysP promoter.

Anticoagulant patient information material is written at high readability levels.

BACKGROUND: Warfarin therapy requires frequent monitoring and dose adjustment. Elderly patients with atrial fibrillation, prior stroke, and lower literacy skills may have difficulty reading brochures that explain dosing instructions, procedures to follow, and the risks and benefits of anticoagulants. In general, it is recommended that brochures be written at or below the 6th-grade level. We determined the readability of patient information material being offered to patients receiving anticoagulants. METHODS AND RESULTS: We used the SMOG grade formula to measure readability of written patient materials. We obtained 50 brochures commonly used in anticoagulation management units from industry and health advocacy groups. Patient information was related to atrial fibrillation (16%, n=8), warfarin (44%, n=22), low-molecular-weight heparins (12%, n=6), or other related topics (28%, n=14). The mean readability was found to be grade 10.7 (95% CI 10.1 to 11.2); none had a readability score at the 6th-grade level or below, 12% of the brochures had readability scores at the 7th- to 8th-grade levels (n=6), 74% at the 9th- to 12th-grade levels (n=37), and 14% at higher than 12th-grade level (n=7). The readability grade level was similar for brochures produced by industry or health advocacy groups (P:=0.9) but higher for information obtained from the Internet (12.2+/-1.3 grades) compared with other sources (10.3+/-2.1 grades; P:=0.01). CONCLUSIONS: Patient education materials related to the use of anticoagulants are written at grade levels beyond the comprehension of most patients. Low-literacy brochures are needed for patients on anticoagulants.

The Escherichia coli ssuEADCB gene cluster is required for the utilization of sulfur from aliphatic sulfonates and is regulated by the transcriptional activator Cbl.

The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH(2)-dependent monooxygenase and an NAD(P)H-dependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639-26646). Synthesis of beta-galactosidase from a transcriptional chromosomal ssuE'-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysR-type transcriptional regulator. Electrophoretic mobility shift assays with the ssu promoter region and measurements of beta-galactosidase from plasmid-encoded ssuE'-'lacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the -35 region. CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE'-lacZ fusion although the ssu promoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE'-lacZ fusion.

Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

Starvation for sulfate results in increased synthesis of several proteins in Escherichia coli. Among these Ssi (sulfate starvation-induced) proteins are the products of the tauABCD genes, which are required for utilization of taurine as sulfur source for growth. In this study, the role of the cbl gene in expression of tauABCD and other ssi genes was investigated. The protein encoded by cbl shows high sequence similarity to CysB, the LysR-type transcriptional activator of the genes involved in cysteine biosynthesis. Strain EC2541, which contains an internal deletion in cbl, was unable to utilize taurine and other aliphatic sulfonates as sulfur sources. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many of the Ssi proteins were not synthesized in EC2541. Expression of a translational tauD'-'lacZ fusion required the presence of both cbl and cysB. The interactions of CysB and Cbl with the promoter region of tauABCD were studied by using gel mobility shift experiments and DNase I footprinting. CysB occupied multiple binding sites, whereas Cbl occupied only one site from 112 to 68 bp upstream of the transcription start site. Acetylserine, the inducer of transcription of CysB-regulated genes, stimulated binding of CysB but not of Cbl. Sulfate had no effect on binding of both proteins to the tauABCD promoter region. These results indicate that Cbl is a transcription factor for genes required for sulfonate-sulfur utilization and maybe for other genes whose expression is induced by sulfate starvation.

A new gene, cbl, encoding a member of the LysR family of transcriptional regulators belongs to Escherichia coli cys regulon.

The cbl (cysB-like) gene has been identified in Escherichia coli. The analysis of the cloned cbl sequence revealed strict homology to an ORF of unknown function found initially in Klebsiella aerogenes [Schwacha and Bender, J. Bacteriol. 175 (1993) 2107-2115]. The predicted Cbl protein has structural features of the LysR family of transcriptional activators. It is also strongly similar to the CysB protein, the activator of the cys regulon. The position of cbl on the Ec physical map has been established at a 2070-kb (43.5 min) region between asnU and asnV. The gene is expressed in vivo as a 1-kb monocistronic transcript starting from one major transcription start point. Unexpectedly, the in vivo expression of cbl has shown dependence on CysB, belonging to the same family of proteins. The promoter region of cbl binds purified CysB protein in a manner similar to other CysB-responsive promoters. A cbl disruption mutant was constructed by insertion of a KmR gene cartridge into the ORF on the chromosome. Phenotypes related to cbl expression suggest the involvement of the gene in an accessory regulatory circuit within the cys regulon engaging, in the last step, the function of the cysM gene encoding O-acetylserine (thiol)-lyase B.

Chromosomal localization and catalytic properties of the recombinant alpha subunit of human lymphocyte methionine adenosyltransferase.

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.

Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium.

CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium.

CysB is a member of the LysR family of transcriptional activators and regulates genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli. CysB binds to specific sites just upstream of the -35 regions of the cysJIH, cysK, and cysP promoters, where, in the presence of N-acetyl-L-serine, it stimulates transcription initiation. The cysK and cysP promoters contain additional binding sites, and we have proposed that CysB bends these promoters by binding to adjacent sites. N-Acetyl-L-serine is thought to decrease the magnitude of such bending. Since stoichiometric data bearing on this model have been lacking, we analyzed complexes in gel mobility shift experiments with 35S-labeled CysB and 32P-labeled promoter fragments. CysB was found to bind as a tetramer, and N-acetyl-L-serine increased the electrophoretic mobilities of one-protein complexes of the multibinding site cysK and cysP promoters without changing their stoichiometry, indicating that a single CysB tetramer can bend these promoters and that N-acetyl-L-serine diminishes such bending. Bend angles for both promoters were calculated to be 100 and 50 degrees in the absence and presence of N-acetyl-L-serine. N-Acetyl-L-serine affected neither the stoichiometry nor the electrophoretic mobility of cysJIH promoter complexes, which are not known to contain bent DNA. DNA bending may be a mechanism for sequestering CysB at certain promoter sites by increasing their affinity for this protein in the absence of N-acetyl-L-serine.

The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate.

The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine, N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind at CBS-P2, giving a more slowly migrating complex.

In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium.

The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchin Strongylocentrotus purpuratus.

The general organization and primary amino acid sequences of the S. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26-27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5'-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5'-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage. The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.

Polyamines enhance readthrough of the UGA termination codon in a mammalian messenger RNA.

The polyamines spermidine and spermine stimulate the readthrough of the UGA termination codon of rabbit beta-globin mRNA when it is translated in a rabbit reticulocyte cell-free system. The other major polyamine, putrescine, does not show this effect. The polyamine induced readthrough is specific for UGA as the UAA termination codon of alpha-globin mRNA is not read through and general translational misreading errors are not occurring in the presence of spermidine or spermine. The probable mechanism of this effect and some possible regulatory implications are discussed.