A Complex of Enzymatically Synthesized Rna and Template Dna

Bremer, H. J.; Konrad, Michael

doi:10.1073/pnas.51.5.801

Cited by 134 publications

(18 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This transcription complex was first described by Bremer and Konrad. 29 Wehrli, Kniisel, and Staehelin22 have reported that rifamycin inhibits RNA polymerase by interacting with the enzyme rather than with the DNA. Sippel and Hartmann23 have presented evidence indicating that enzyme molecules in the form of either the initiation or transcription complex are refractory to the action of this inhibitor.…”

mentioning

confidence: 99%

TRANSIENT ACTIVATION OF RNA POLYMERASE IN Escherichia coli B AFTER INFECTION WITH BACTERIOPHAGE T4

Oleson¹,

Pispa²,

Buchanan³

1969

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Abtract.-Sk6ld and Buchanan' have reported that there is a rapid loss of RNA polymerase activity in Escherichia coli B after infection with T4 bacteriophage. More recent studies on the mechanism of this inactivation have been made in this2 and other laboratoriesA 34 In this communication, we report the observation of a transient stimulation of RNA polymerase activity when measurement is made immediately after infection and when cells are ruptured by a gentle lysis procedure. The increase in activity is independent of the synthesis of protein.The activity in the extracts of infected cells is lost by treatment of the extract with antibody to E. coli RNA polymerase and is refractive to the inhibitory action of the antibiotic rifamycin. Hybridization experiments indicate that an RNA transcribed almost exclusively from a T4 DNA template is the product of incubation of extracts of infected cells with a reaction mixture containing an exogenous primer (salmon sperm DNA). These findings are consistent with the hypothesis that one of the first steps in phage infection is the formation of a transcription complex containing T4 DNA and E. coli RNA polymerase.Experimental Procedure.-Materials: The following materials were purchased from the indicated suppliers: salmon sperm DNA, Calbiochem, Inc.; unlabeled nucleoside triphosphates, P-L Biochemicals, Inc.; "4C-UTP, Nuclear-Chicago Corp.; 3H-UTP, a-32P-UTP, and '4C-dGMP, Schwarz BioResearch, Inc.; pronase, bovine serum albumin, and spermidine -3 HC1, Sigma Chemical Co.; creatine phosphokinase and creatine phosphate, Boehringer; Liquifluor and '4C--leucine, New England Nuclear Corp.; pancreatic DNase and egg white lysozyme, Worthington Biochemical Corp. Chloramphenicol and rifamycin B were gifts of Parke, Davis and Co. and Ciba Pharmaceuticals, respectively. DNA from E. coli B was prepared by a modification of the procedure of Marmur.9 T4 DNA was prepared by the procedure of Kaiser and Hogness.6 RNA polymerase was purified by the method of Babinet7 from E. coli B. The serum globulin fraction from nonimmune rabbits and from rabbits immunized against purified RNA polymerase was prepared by collection of the protein precipitating from 0-50% saturation with ammonium sulfate. Bacteriophage ghosts, prepared from T4 phage by osmotic shock,8 were separated from residual unbroken phage by centrifugation in a discontinuous gradient' of CsCl.Analytical procedures: Standard methods were used to determine the concentration of bacteria and of bacteriophage.10 Titers of bacteriophage ghosts were estimated by determination of their serum blocking power." The concentration of protein in crude extracts was measured by a biuret method'2 after precipitation with trichloroacetic acid. The concentration of DNA was measured by its absorbance at 260 m/A. A value of 20 was used as the absorbance of a 0.1% solution.Preparation of extracts: Two procedures, both utilizing lysozyme as a means of disrupting cell walls of E. coli B, are described in the legend of Table 1. Procedure 2, which did not include u...

show abstract

mentioning

confidence: 99%

TRANSIENT ACTIVATION OF RNA POLYMERASE IN Escherichia coli B AFTER INFECTION WITH BACTERIOPHAGE T4

Oleson¹,

Pispa²,

Buchanan³

1969

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Incorporation of -y-32p-ATP and y-32P-GTP were determined separately and the results added. Incubation was at 370 with 3.3 units of enzyme (equivalent to 3 1s&moles of polymerase) and 12 The conditions of the assay were the same as those for Table 1 except that rifampicin (final concentration 0.5 pg/ml) was added at the time indicated in the table.…”

mentioning

confidence: 99%

DNA-DEPENDENT SYNTHESIS OF RNA BY Escherichia coli RNA POLYMERASE: RELEASE AND REINITIATION OF RNA CHAINS FROM DNA TEMPLATES

Maitra

Barash

1969

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Abstract.-RNA synthesis in an in vitro RNA polymerase system at low ionic strength soon ceases, due to inhibition by accumulated RNA. Measurement of RNA chain initiation by the incorporation of y_-32P-ATP and GTP with native T2 or T4 DNA as template shows that only one RNA chain is formed per molecule of enzyme added. In contrast, when the polymerase reaction is carried out in 10 mM Mg++ and 0.2 M KCl, RNA synthesis proceeds nearly linearly for hours, resulting in a marked increase in accumulated RNA. Incorporation of y-32P-ATP also proceeds throughout the course of the reaction and the number of chains initiated per molecule of enzyme is increased severalfold. Most RNA chains formed are released from the DNA template as free RNA. Polymerase is released also in this process and acting catalytically reinitiates new chains. Rifampicin inhibits initiation of RNA synthesis and also blocks reinitiation of RNA chains without affecting growth of RNA chains already initiated.Introduction.-The DNA-dependent RNA polymerase reaction in vitro can be divided into a number of discrete steps. These include (a) recognition by the polymerase of specific starting points on the template DNA at which enzyme molecules bind to DNA and (b) initiation of RNA chains. It has been shown previouslyl-4 that the initiation reaction, measured by the incorporation of

show abstract

“…Further kinetic analyses, including "pulse-chase" experiments (29) and "actinomycin chase" experiments (30), will be needed to clarify this point. Ribosomes may have a role in regulating the synthesis of RNA and its release from DNA template (31)(32)(33)(34). Hence, it would be attractive to postulate that polysomes are formed in the nucleus.…”

mentioning

confidence: 99%

Isolation of Two Distinct Classes of Polysomes From a Nuclear Fraction of Rat Liver

Sadowski

Howden

1968

The Journal of Cell Biology

View full text Add to dashboard Cite

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-' 4 C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-' 4 C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.

show abstract

A Complex of Enzymatically Synthesized Rna and Template Dna

Cited by 134 publications

References 12 publications

TRANSIENT ACTIVATION OF RNA POLYMERASE IN Escherichia coli B AFTER INFECTION WITH BACTERIOPHAGE T4

TRANSIENT ACTIVATION OF RNA POLYMERASE IN Escherichia coli B AFTER INFECTION WITH BACTERIOPHAGE T4

DNA-DEPENDENT SYNTHESIS OF RNA BY Escherichia coli RNA POLYMERASE: RELEASE AND REINITIATION OF RNA CHAINS FROM DNA TEMPLATES

Isolation of Two Distinct Classes of Polysomes From a Nuclear Fraction of Rat Liver

Contact Info

Product

Resources

About