Biomedical subjects
Publications and source records attributed to H Zalkin.
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The Bacillus subtilis pur operon is a 12-gene cluster, purEKB-purC(orf)QLF-purMNH(J)-purD, organized in groups of overlapping coding units separated by intercistronic gaps. Translational fusions of Escherichia coli lacZ were constructed to purE, purC, and purM, the first gene of each group. Analyses of gene fusions integrated into the chromosomal pur operon exclude the possibility of internal promoters in intercistronic regions and support the view that transcription is from the single sigma 43 promoter at the 5' end of the operon. Enzyme and mRNA measurements indicate that transcriptional regulation occurs solely at the 5' end of the operon. The relative levels of beta-galactosidase from purE-lacZ, purC-lacZ, and purM-lacZ were determined under repressing and nonrepressing conditions. These results indicate that expression of purC-lacZ was 3.0- to 6.8-fold higher than purE-lacZ because of enhanced translational efficiency. The enhanced translational efficiency of purC-lacZ was accompanied by a partial escape from regulation by purines. This anomalous effect on purC-lacZ was the only suggestion for posttranscriptional regulation.
Mutations were constructed in vitro which identify the -35 promoter element and the operator site of the Escherichia coli purF operon as well as confirm the -10 promoter sequence. The operator was localized by a two-base change at positions -26 and -27, relative to the start of transcription. This mutation abolished repression of a purF-lacZ fusion. In the wild-type, repression of single copy and multicopy purF-lacZ constructs was equally effective. This indicates that cells contain a greater than 100-fold excess of purR-encoded repressor than is needed to regulate the chromosomal purF operon. Thus, cells contain sufficient repressor to regulate the other pur regulon genes. Two independent purR mutations were isolated which abolished repression of purF and purF-lacZ. We conclude that there is a single repressor protein-operator regulatory system to sense purine or purine nucleotide pools.
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.
Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine nucleotides (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide, 5'-untranslated mRNA leader region. We have identified an apparently intact, terminated transcript of approximately 200 nucleotides in length, having a half-life of about 0.7 min. The terminated transcript is degraded in a series of discrete steps resulting in the accumulation of stable intermediates in vivo. We have used Northern blot analysis, primer extension, and nuclease S1 mapping to align the degradation intermediates with the nucleotide sequence and assign secondary structures that may contribute to the stability of the intermediates. Degradation is initiated by endonucleolytic cleavage of the approximately 200-nucleotide terminated transcript generating approximately 93- and approximately 97-nucleotide 5' and 3' moieties, respectively. The approximately 93-nucleotide 5' and approximately 97-nucleotide 3' intermediates are further degraded to approximately 88 and approximately 58 nucleotides, respectively. The 5'-end of pur operon mRNA and the attenuated transcript are degraded by different pathways.
Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region. These mutations affect processing and enzyme activities. Processing of the leader is dependent upon glutamic acid residues at positions -2 and -1 as well as Cys1. In addition, processing requires a pro-peptide longer than 3 residues. Function of the active site cysteine is dependent on pro-peptide processing. Enzyme purified from a pro-peptide deletion strain has activity and iron content that is comparable to the wild type. These results establish that the pro-peptide is not essential for enzyme maturation, but they leave unanswered the question of pro-peptide function.
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In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.
An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P. J., and Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179) was cloned. The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP. The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes. The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila. Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B. subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes. S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA. These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.
Yeast genes under general amino acid control contain multiple copies of a sequence known as the TGACT repeat in the 5'-flanking DNA. The yeast TRP5 gene contains two copies of the TGACT repeat sequence in its 5'-flanking region. The upstream TGACT repeat of TRP5 is required for normal basal expression as well as derepression by general control. Synthetic oligonucleotides containing a TGACT sequence were inserted into previously constructed TRP5 control region deletion mutants. A synthetic 17-base pairs (bp) oligonucleotide containing a TGACT copy along with flanking nucleotides from HIS3 was able to restore derepression in all deletion mutants tested. The 17-bp oligonucleotide also functioned bidirectionally. Replacements in the upstream control region by synthetic oligonucleotides indicated that sequences other than the TGACT repeat are required for high basal expression. Replacements of the downstream repeat sequence by the 17-bp oligonucleotide suggest its main role in this position is for derepressed expression. High level derepressed expression was found to correlate with the presence of two repeats.
Site-directed mutations were introduced into a conserved region of the Escherichia coli CTP synthetase glutamine amide transfer domain. The amino acid replacements, valine 349 to serine, glycine 351 to alanine, glycine 352 to proline, and glycine 352 to cysteine, all increased the lability of CTP synthetase. The proline 352 replacement abolished the capacity to form the covalent glutaminyl-cysteine 379 catalytic intermediate, thus preventing glutamine amide transfer function; NH3-dependent CTP synthetase activity was retained. In CTP synthetase (serine 349), both glutamine and NH3-dependent activities were increased approximately 30% relative to that of the wild type. CTP synthetase mutants alanine 351 and cysteine 352 were not overproduced because of apparent instability and proteolytic degradation. We conclude that the conserved region between residues 346 and 355 in the CTP synthetase glutamine amide transfer domain has an important structural role.
Several mutations were constructed in residues thought to provide ligands for a [4Fe-4S] cluster in Bacillus subtilis amidophosphoribosyltransferase using site-directed mutagenesis of cloned purF. These replacements confirm the identification of cysteinyl ligands to the Fe-S center. Of five mutant enzymes, two had no activity, two less than 25% of the wild type activity, and one was lethal and could not be studied. The Fe content of the two mutant enzymes with partial activity was similar to that of the wild type. Results of partial characterization suggest that the [4Fe-4S] cluster is not involved in allosteric regulation and does not play a specific role in the ammonia- or glutamine-dependent reactions of the enzyme. At least partial enzymatic activity is required for NH2-terminal processing. Pulse labeling experiments suggest that processing is a slow post-translational process which is dependent upon cellular factors. A relationship between Fe-S centers and NH2-terminal processing of an undecapeptide leader suggests a functional connection between these two structural elements in amidophosphoribosyltransferase.
The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.
Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.
Escherichia coli purF has been determined to be the distal gene of a polycistronic operon. The first gene of the purF operon encodes a hydrophobic 17.9-kDa protein of unknown function. Deletion analyses indicate that the 17.9-kDa protein plays no role in the regulation of purF in cis. mRNA hybridization studies establish that purF is regulated at the transcriptional level. Enzyme and mRNA levels are repressed 11-17-fold by excess adenine. A single mRNA start site at nucleotide +1 was identified for transcripts synthesized in vivo. Two sites, at +1 and approximately +30, were used for transcription initiation in vitro. The purF promoter is localized between nucleotides -96 and -7 with sequences upstream of -71 necessary for high level expression. Initial evidence suggests that transcription is subject to stringent control. Deletion analyses localize the purF control element to a region between nucleotides -71 and +35. A putative control site between nucleotides -35 to +3 strongly resembles a 5' flanking sequence in the co-regulated gene purM. This site contains an imperfect inverted repeat sequence that is characteristic of sites recognized by regulatory proteins and is a candidate for the purF operator. This is the first detailed analysis of a gene involved in de novo purine nucleotide biosynthesis.
GMP synthetase (EC 6.3.4.1), a glutamine amido-transferase encoded by the guaA gene, catalyzes the synthesis of GMP from XMP. The guaA gene was subcloned from the Clarke and Carbon (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) plasmid pLC34-10, and the nucleotide sequence was determined. The structural gene encodes a protein of 525 amino acid residues having a calculated Mr of 58,604. The amino acid sequence of the NH2 terminus of GMP synthetase was determined and used to verify the translation start site determined from the DNA sequence. A 68-base pair intercistronic region separates guaA from the upstream guaB gene in the polycistronic guaBA operon. The 3' end of the guaA mRNA was determined by S1 nuclease mapping. The 3' end of guaA mRNA is 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a rho-independent transcription termination site.
The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.
The yeast gene TRP5 is regulated by the general control system of amino acid biosynthesis. A TRP5-lacZ translational fusion was constructed in order to facilitate assay for TRP5 promoter function and regulation. The chromosomally integrated fusion was derepressed 4-5-fold by lysine limitation. Thus, the TRP5-lacZ fusion is regulated by general control. Deletions were constructed in vitro in the 5'-flanking region of cloned TRP5-lacZ. These deletions localized the promoter region to within 188 base pairs of the transcription start site. Two separable subregions of the promoter were localized by further deletion mapping. The boundaries of the promoter regions are approximately -188 to -114 and -91 to -29. The TATA box is contained within the downstream promoter region. Regulatory regions essential for general control overlap with these promoter elements. Each of two separable regulatory regions contains a copy of the TGACT repeat (Donahue, T. F., Daves, R. S., Lucchini, G., and Fink, G. R. (1983) Cell 32, 89-98) previously implicated in regulation of HIS4 by general control.