Organization of transfer and ribosomal ribonucleic acid genes in Bacillus subtilis.

The structural relationship between the transfer ribonucleic acid (tRNA) and the ribosomal RNA (rRNA) genes of Bacillus subtilis has been studied by restriction endonuclease analysis of total chromosomal deoxyribonucleic acid (DNA) and characterization of DNA fragments cloned in Escherichia coli. The DNA sequences encoding rRNA and tRNA were assayed by hybridization to radioactive RNA. The results support the conclusion that the tRNA genes are interspersed between and closely linked to the rRNA genes of B. subtilis. They probably do not appear between the 16S and 23S rRNA genes as in E. coli.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

Stability of ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

After heating at 65 C, ribosomes isolated from Bacillus stearothermophilus were strikingly more heat-stable than comparable preparations from Escherichia coli when tested for ability to support polyuridylic acid-directed phenylalanine incorporation at 37 C. The stability of ribosomes was also determined by measurements of hyperchromicity at 259 mmu while heating them from 25 to 90 C. In standard buffer containing 0.01 m Mg(++), the T(m) (temperature at the midpoint of total hyperchromicity) of E. coli and B. stearothermophilus ribosomes was 71 and 81 C, respectively. In a magnesium-free buffer, the T(m) of E. coli and B. stearothermophilus ribosomes was 44 and 64 C, respectively. Putrescine (0.01 m) was more effective in stabilizing ribosomes from B. stearothermophilus than those from E. coli. Spermidine (0.001 m), on the other hand, was more effective in stabilizing ribosomes from E. coli than those from B. stearothermophilus. Melting curves of total ribosomal ribonucleic acid (rRNA) from E. coli and B. stearothermophilus revealed T(m) values of 50 and 60 C, respectively. Putrescine stabilized thermophile rRNA, but had no effect on E. coli rRNA. Sucrose density gradients demonstrated that thermophile 23S ribonucleic acid was degraded during storage at -20 C; the 23S component from E. coli was stable under these conditions. The results are discussed in terms of the mechanism of ribosome heat stability and the role of the ribosome in governing the temperature limits for bacterial growth.     

Inhibition of Histoplasma capsulatum ribonucleic acid polymerases by homologous and heterologous ribonucleic acid.

The ribonucleic acid (RNA) polymerases from the yeast phase of Histoplasma capsulatum are differentially sensitive to RNA isolated from the yeast and mycelial phases of this fungus and from Escherichia coli. Low-molecular-weight RNA from H. capsulatum was the most effective inhibitor.     

Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli.

The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity.     

Identification of a temperature-sensitive asparaginyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

A temperature-sensitive mutant of Escherichia coli K-12 isolated previously (H. Ohsawa and B. Maruo, J. Bacteriol. 127:1157-1166, 1976) was found to have an alteration in asparaginyl-transfer ribonucleic acid synthetase. This alteration can account for the temperature-sensitive phenotype of the mutant. No evidence was obtained to support the previous suggestion that ribosomal protein S1 is altered in this mutant. Combined with the previous genetic studies, we conclude that the newly defined genetic locus, asnS, for the asparaginyl-transfer ribonucleic acid synthetase maps near pyrD at 21 min on the E. coli chromosome.     

Potentiation of hemoglobin messenger ribonucleic acid. A step in protein synthesis initiation involving interaction of messenger with 18 S ribosomal ribonucleic acid.

The polyadenylic acid-containing messenger ribonucleic acid from rabbit reticulocyte polyribosomes, isolated by a rapid and very gentle procedure (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084), sediments in a sucrose gradient in three sharp peaks, at 9 S, 17 to 18 S, and 28 S. The alpha and beta globin messenger activity follows the absorbance profile in the sucrose gradients and has its major peak at 17 to 18 S. The larger messengers are more active than 9 S messenger by approximately 2-fold per mass unit of ribonucleic acid or by at least 8-fold per molecule. The major 17 to 18 S form of globin messenger was examined further and was shown to be a 1:1 complex of 9 S messenger and 18 S ribosomal ribonucleic acid. The effect of 18 S ribosomal ribonucleic acid on translation of purified 9 S globin messenger was analyzed in a messenger-dependent protein-synthesizing system (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084). In the absence of exogenous ribosomal ribonucleic acid, 9 S messenger is inefficiently translated; a large excess of messenger is required to saturate the system; and globin is synthesized mainly on di- and monoribosomes. Exogenous liver or reticulocyte 18 S ribosomal ribonucleic acid potentiates 9 S messenger translation and renders it at least 10 times more efficient. The potentiation reaction can also be accomplished by increasing the concentration of ribosomes in the assay system. However, transfer or messenger ribonucleic acids cannot carry out this reaction. It is proposed that 9 S globin messenger ribonucleic acid is an inactive molecule which is normally potentiated by specific reversible base pairing with an accessible region of ribosomal ribonucleic acid contained in a 40 S ribosomal subunit. The potentiated messenger interacts with initiation factors and with other ribosomal subunits to synthesize protein. Potentiation is the first specific function in protein synthesis demonstrated for the ribosomal ribonucleic acid portion of ribosomes.     

Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.     

Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.     

Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms.

The cross-reaction of phenylalanyl-transfer ribonucleic acid (tRNA) ligases from different microorganisms with antibodies raised against the purified enzyme from Escherichia coli has been investigated. The results of immunotitration and immunodiffusion experiments and of the sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of immunoprecipitates revealed: (i) a high degree of immunochemical identity of this enzyme only within the family Enterobacteriaceae; (ii) intermediate-to-weak cross-reaction with the phenylalanyl-tRNA ligases from Pseudomonadaceae, Rhodopseudomonas spheroides, and Bacillus stearothermophilus; (iii) no detectable cross-reaction (with the methods employed) with the enzymes from several gram-positive organisms, Euglena gracilis, and several fungi. As revealed by immunochemical analysis, a merodiploid strain of E. coli carrying an episome (F148) that covers the aroD region of the E. coli chromosome possesses at least twice the amount of phenylalanyl-tRNA ligase in comparison with its haploid parent strain. This suggests that the cistrons for both the alpha and beta polypeptides of this enzyme are mapping in this area.     

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.     

Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization.

With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels. A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection. The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.     

Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase.

Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both. The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid. There is a promoter site before gene A, which lies either in gene H or between H and A. There is evidence for a promotor between genes C and D or in gene C. There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F.     

Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase.

A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.     

Gene Dosage for 5S Ribosomal Ribonucleic Acid in Escherichia coli and Bacillus megaterium

The number of gene copies for 5S ribosomal ribonucleic acid (rRNA), relative to that for 16 and 23S rRNA, has been determined by deoxyribonucleic acid (DNA)-RNA hybridization for Escherichia coli and Bacillus megaterium. In both cases, the number of 5S rRNA genes equals the number of 16 or 23S rRNA genes. Rapid procedures for preparing extremely highly purified DNA suitable for DNA-RNA hybridization experiments and chemically pure 5S rRNA are described.     

Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase.

Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically. The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it. The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction. Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements. Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.