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Sigma factor is not released during transcription in Bacillus subtilis.

The relationship between sigma (sigma) and delta (delta) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When core enzyme (E) containing delta factor (E delta) binds to DNA, the delta factor is released with the formation of an E-DNA complex. The addition of sigma to the E-DNA complex results in the formation of a stable E sigma-DNA complex which can synthesize RNA upon addition of nucleoside triphosphates. Sigma factor, significantly, is not released from the core during RNA synthesis. These results suggest that delta and sigma factors can act sequentially during initiation of RNA synthesis with delta acting as a DNA recognition factor and sigma acting as an initiation factor. The results do not preclude the possibility that E sigma can initiate RNA synthesis correctly since E sigma alone can bind to DNA and initiate RNA synthesis.

Bacillus subtilis

Delta factor can displace sigma factor from Bacillus subtilis RNA polymerase holoenzyme and regulate its initiation activity.

A protein with a molecular weight of 21,000 daltons is found associated with a fraction of Bacillus subtilis RNA polymerase core. This protein (delta) does not react with antibody made against sigma factor and has a peptide map which is significantly different from sigma factor. At ratios of 2:1 to 4:1 (delta:holoenzyme) the delta displaces sigma factor completely from the core and associates in a 1:1 ratio with core to form delta-core. Under the same incubation conditions sigma factor at a ratio of 10:1 (sigma factor:delta-core) does not displace delta from the delta-core. The delta-core has much less activity as compared to holoenzyme on various DNA templates. However, sigma factor does stimulate the activity of delta-core enzyme under conditions of RNA synthesis. These observations and the results of others suggest that delta-core enzyme binds initially to specific DNA sites followed by delta release from the core-DNA complex and that the sigma factor binds to the core-DNA complex to initiate RNA synthesis. Thus both delta and sigma factors are required in a sequential fashion for specific transcription to occur in B subtilis.

Bacillus subtilis

Chromosomal location of a structural gene for the RNA polymerase sigma factor in Escherichia coli.

A set of F' strains of Escherichia coli K-12 partially diploid for various chromosomal segments has been examined for possible gene dosage effects in the synthesis of sigma factor of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate:RNA nucleoti-dyltransferase, EC 2.7.7.6). It was found that all F' strains diploid for the dnaG region synthesize sigma at rates two to three times higher than other F' or F- strains. Moreover, strains of Salmonella typhimurium harboring these F' plasmids produce E. coli sigma in addition to Salmonella sigma. This has been shown on the basis of the finding that Salmonella sigma can be precipitated with antiserum against E. coli RNA polymerase but is distinguishable from E. coli sigma in its mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. E. coli sigma polypeptides thus produced seem to be stable in cells of S. typhimurium. These results indicate that a structural gene for sigma (rpoD) is located at the metC-argG region, probably near the dnaG locus (66 min on the current genetic map of E. coli).

Alleles

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.

Amino Acid Sequence

Isolation and characterization of transducing phage coding for sigma subunit of Escherichia coli RNA polymerase.

A transducing phage has been isolated with codes for the sigma subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage lambda vector that is capble of forming lyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the sigma gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and sigma (rpoD) whereas those containing the Sac I fragment complement only sigma. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/translation system suggest that a Sac I site separates the promoter for sigma from the sigma structural gene. The direction of transcription of sigma was determined to be clockwise with respect to the E. coli genetic map.

Bacterial Proteins

Three methods to elicit sigma-optokinetic nystagmus in Java monkeys.

Sigma-optokinetic nystagmus (sigma-OKN) can be elicited in awake Java monkeys (Macaca fascicularis) when stationary periodic visual patterns (grid of black white stripes, row of equally spaced dots) are illuminated stroboscopically. Three methods were found to be useful in inducing the sigma-OKN: postrotatory nystagmus, optokinetic afternystagmus (OKAN) following normal OKN and a gradual transition from phi-movement (phi-OKN) to sigma-OKN. The properties found for sigma-OKN in man are also present in monkeys with the one exception that monkeys have a long-lasting sigma-OKAN in darkness which is not present in man. The average angular speed Ve of sigma-OKN slow phases was related to the flash frequency fs and the spatial period Ps of the stripe pattern according to the following equation: Ve = k.Ps . fs [degrees . s-1] The constant k was 1 or close to 1.

Animals

Altered chemical properties in three mutants of E. coli RNA polymerase sigma subunit.

We have analyzed some chemical properties of the sigma subunit of RNA polymerase from the sigma mutants: rpoD1 (Gross et al., 1978), rpoD2 (formerly known as alt-1) (Silverstone et al., 1972; Travers et al., 1978), and rpoD800 (Gross et al., 1979). Each of the three mutants is located at about 66 min on the E. coli genetic map and exhibits an alteration in the enzymatic properties of its sigma subunit. The tryptic peptides and isoelectric focusing behavior were analyzed for mutant and wild type sigma. A single, but different altered lysine tryptic peptide was observed for each mutant. No altered arginine tryptic peptides were observed. The rpoD800 mutant sigma showed an altered isoelectric point. These studies provide chemical evidence that the sigma polypeptide in all three mutants is altered and strongly support the conclusion that the mutations are in the structural gene for sigma.

Alleles

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

A gene affecting the sigma subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 min, and the order tolC-dnaG-sigma-tet was determined. We used frequency of contransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the sigma subunit. One such mutant was found to map at the sigma locus. The sigma subunit isolated from this mutant is unstable at 46 degrees C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is sigma dependent.

Chromosome Mapping

Mutation affecting thermostability of sigma subunit of Escherichia coli RNA polymerase lies near the dnaG locus at about 66 min on the E. coli genetic map.

The Escherichia coli strain, ts-rnp5, originally described in 1975 by G. D. Burdick and H. Berger, is shown to possess an RNA polymerase (RNA nucleotidyltransferase) sigma subunit with an activity 4--6 times less thermostable at 45 degrees than sigma from wild-type strains. This defect remains associated with the sigma polypeptide through a variety of purification stages, including renaturation of sigma after its elution from sodium dodecyl sulfate/polyacrylamide gels. The mutation responsible for decreased thermostability of sigma, called rpoD1, cotransduces with dnaG and therefore is located at about 66 min of the E. coli genetic map.

Chromosome Mapping

Sigma subunit of Escherichia coli RNA polymerase affects the function of lambda N gene.

A new class of Escherichia coli mutants, referred to as grn, has been isolated by localized mutagenesis. These mutations affect the sigma subunit of DNA-dependent RNA polymerase (ribonucleoside 5'-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) by abolishing the expression of the lambda N gene, and they are closely lniked to dnaG in the order dnaG-grn-uxaA. Detailed study of one such mutant, grn1, yielded the following results: (i) grn1 is a single mutation and the mutant cell shows cold-sensitivity in growth; (ii) the Grn phenotype of the mutant can easily be suppressed by secondary mutations in the beta subunit gene of RNA polymerase; (iii) purified holoenzyme of RNA polymerase isolated from the mutant showed an altered salt-dependency in vitro, and the mixed reconstitution of the mutant with the wild-type subunits showed that the sigma subunit of the grn1 mutant is altered; (iv) lambda phage mutants (lambda grg), which overcome the grn mutation, can be classified into two groups, the "nin-deletion" and the "N-mutant" groups (both of these are also able to grow on the previously described groN mutant of Georgopoulos and nusAB of Friedman); (iv) the mutant polymerase transcribed 12S as well as 7S RNA from lambda DNA in the presence of the rho factor in vitro. These results indicate that the grn mutation alters the sigma subunit of RNA polymerase and that the sigma subunit participates in activating the N-mediated antitermination mode of lambda phage transcription.

Bacteriophage lambda

The stability of the sigma sleep spindle.

The present study closely examined the distribution of sigma sleep spindle activity or six normal human males who slept undisturbed for approximately 8 h/night for 3 consecutive nights. Sigma spindle activity was monitored by an automatic spindle detector system which performed at 92.5% accuracy when judged against established visual criteria. Individuals differed markedly in total sigma spindle production across nights, but the failure to detect significant differences among nights and the large intra-class correlation suggests noteworthy inter-night stability of the sigma spindle.

Adult

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toIC and dnaG, at 66 min.

Chromosome Mapping

Distinctive nucleotide sequences of promoters recognized by RNA polymerase containing a phage-coded "sigma-like" protein.

We report the nucleotide sequences of two promoters for bacteriophage SP01 "middle" genes. These promoters are recognized by a modified form of Bacillus subtilis RNA polymerase that contains a phage-coded "sigma-like" regulatory protein (gp28) in place of the bacterial sigma factor. Both promoters shared the identical hexanucleotide 5'A-G-G-A-G-A at about 35 base pairs preceding the start point of transcription and the identical heptanucleotide 5'-T-T-T-A-T-T-T (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA) located about 10 base pairs preceding the transcriptional start point. The significance of these sequences in comparison with nucleotide sequences of promoters recognized by sigma-containing RNA polymerases is discussed.

Bacillus subtilis

Nitroglycerin in acute myocardial infarction. X. Effect of small and large doses of nitroglycerin on sigma ST segment deviation -- experimental and clinical results.

The purpose of the present study was to investigate the effect of the dose of nitroglycerin (NTG) on myocardial ischemic injury. In 20 closed chest dogs the anterior descending branch of the left coronary artery was occluded by inflating a balloon in its lumen. Compared with the untreated control group the sigma ST elevation was significantly lower when NTG was applied at a rate of 0.02 mg/min, but significantly higher when NTG was administered at a rate of 0.10 mg/min. In 12 patients with acute myocardial infarction NTG was infused at a rate of 3 mg in the first hour (0.05 mg/min) and 6 mg in the second hour (0.1 mg/min). Sigma ST elevation and sigma ST depression decreased during the lower infusion rate (p less than 0.001). When the rate of NTG infusion was raised to 6 mg/hr, the improvement in ST segment deviation was partially reversed. This effect, particularly evident in patients not in heart failure, was associated with a significant rise in heart rate (p less than 0.05) and a fall in diastolic arterial pressure (p less than 0.025). Patients with left ventricular failure were less sensitive to higher doses of NTG than those without failure. Thus, the effect of NTG on myocardial ischemic injury depends on the NTG dose and on the functional state of the injured left ventricle.

Animals

Over-synthesis and instability of sigma protein in a merodiploid strain of Escherichia coli.

We have used two different methods to study the rates of RNA polymerase subunit synthesis in haploid Escherichia coli K12, and a KLF10 rPOB, C+ merodiploid derivative, when grown in glucose-minimal medium at 37 degrees C. Our results indicate that the haploid strain produces beta, beta', alpha and sigma in the molar ratios 1.01:0.99: less than or equal to 2.90:0.26; and that all these subunits are reasonably stable during subsequent growth. The merodiploid produces alpha at the same rate as the haploid, beta and beta' at a 42% higher rate, and sigma at twice the rate. Some 40% of the newly synthesised beta and beta' is degraded within one hour; the residuum is as stable as in the haploid. Alpha is stable throughout. By contrast, sigma is subject to a marked and continuous turnover in the merodiploid. These results are discussed in terms of gene dosage and regulatory effects.

Chromosomes, Bacterial

RNA polymerase mutant with altered sigma factor in Escherichia coli.

A structural gene for sigma factor (rpoD) of DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate: RNA nucleotidyltransferase, E.C. 2.7.7.6) was mapped precisely by a set of F' factors including those already published (Proc. Natl. Acad. Sci. USA. 74, 1831-1835 (1977)). Based on the result that rpoD is located at the dnaG-uxaAC region, a number of mutants containing a temperature-sensitive mutation at or near the uxaA gene were isolated by localized mutagenesis. One of these mutants was found to produce RNA polymerase altered in both thermostability and optimum salt concentration as a result of structural alteration of sigma factor. This mutation, U303, maps at 66 min on the genetic map of E. coli, near the dnaG locus, and affects normal growth of cells.

Chromosome Mapping

The dissociation of sigma-factor from ribonucleic acid polymerase.

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.

DNA-Directed RNA Polymerases

Sigma effects of nalorphine in the chronic spinal dog.

The effects of graded doses of nalorphine and morphine were studied in nondependent chronic spinal dogs. Morphine and low doses of nalorphine produced behavioral changes characterized by indifference, whereas the largest dose of nalorphine produced canine delirium indistinguishable from that produced by SKF-10, 047 or cyclazocine. Nalorphine depressed the flexor reflex; however, a plateau was observed. The data suggest that nalorphine is a partial agonist of the kappa type and a sigma agonist in addition to being a competitive antagonist at the mu receptor, and further, that the dysphoric and hallucinogenic effects of nalorphine-like drugs are due to their sigma activity.

Animals