New Inhibitors of RNA Synthesis <i>in vitro</i> Isolated from <i>Escherichia coli</i>

Kagawa, Haruo; Egberts, E.

doi:10.1111/j.1432-1033.1974.tb03834.x

Cited by 3 publications

(1 citation statement)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since the inhibitor appears to act at the termination step in such a way as to prevent further initiation, it has some features in common with other proteins known to affect termination such as rho [4], kappa [22] and I [23]. All of these proteins depress the total amount of RNA synthesized and show qualitatively similar kinetics of action.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

Crimaldi

Ihler

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

An inhibitor of the Escherichia coli RNA polymerase has been isolated from E. coli and has been partially characterized. The inhibitor, a polypeptide of molecular weight 70000, acts to shut off RNA synthesis at about the time that the first round of RNA synthesis is over, preventing any further RNA synthesis. The inhibitor apparently does not recognize specific termination sequences in the DNA template, since it works equally well with double-stranded DNA, single-stranded DNA and poly[d(A-T)] as templates for RNA synthesis, and because the RNA molecules synthesized from T7 DNA appear to be terminated at the same site either in the presence or absence of the inhibitor. Several experiments indirectly indicate that the inhibitor may reversibly bind to the RNA polymerase at the termination step, in a ratio of approximately one inhibitor molecule per polymerase molecule.The core RNA polymerase of Escherichia coli consist of three different polypeptides, p' (molecular weight 165000), p (molecular weight 155000) and M (molecular weight 39 000) with the subunit composition pP'a2 [1,2]. Core polymerase is the minimum form of the enzyme capable of polymerization of ribonucleoside triphosphates into RNA, but a variety of other polypeptides are known regulate or modify the specificity and activity of core enzyme, Among these are polypeptides which play a role in initiation, such as sigma and the adenosine 3' : 5'-monophosphate acceptor protein and others which play a role in termination, such as rho, as well as various relatively uncharacterized stimulatory and inhibitory proteins. (For a recent review of RNA synthesis, see [3].) Rho factor [4] is required for efficient termination of RNA synthesis at specific sites on the DNA and helps regulate the amounts and kinds of RNA species synthesized. We have isolated a new polypeptide which has some features in common with rho, but which does not appear to recognize specific sites on the DNA. It may play a role in regulating the amount of active RNA polymerase. Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6). MATERIALS AND METHODS Purification10 g casamino acids, 6 g NazHP04 . 7 H20, 3.2 g KH2P04, 10 ml glycerol and 0.3 mg MgS04. Cell growth was stopped at a cell density of 6 x lo8 cells/ ml by the addition of ice. The cells were harvested using a DeLaval cream separator and frozen at -20 "C. 50-60 g of frozen cells, were mixed with 100 g of hydrochloric-acid-washed glass beads (3M Co. Superbrite 100) and 50 ml of grinding buffer (0.05 M Tris pH 7.9,0.25 M KC1,O. 1 M dithiothreitol, 0.1 mM EDTA and 5 % glycerol) and were homogenized in a solid COz/acetone/water-cooled Waring blender for 5 min at low speed and 10 min at high speed. DNase I (0.5 mg) was added and the supernatant decanted after 30 min at 4 "C. The glass beads were reextracted with the addition of 25 ml of grinding buffer. Debris and ribosomes were removed by centrifugation for 2 h at 30000 rev./min in a Spinco 30 rotor and the supernatant was fractionated by the addition of solid (N&)zS04. The material which preci...

show abstract

Section: Discussionmentioning

confidence: 99%

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

Crimaldi

Ihler

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

RNA Polymerase of Escherichia coli

Lathe

1978

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Effect of insect hormones on RNA polymerases of mass-isolated imaginal discs of Drosophila melanogaster cultured in vitro.

Nishiura¹,

Fristrom²

1975

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

View full text Add to dashboard Cite

Four chromatographically separable DNAdependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) were partially purified from imaginal discs of Drosophila melanogaster. Their properties are similar to those described for RNA polymerases I and II isolated from other eukaryotes. In vitro incubation of discs with P-ecdysone, juvenile hormone, or cycloheximide resulted in increased activity of RNA polymerase I. The increase was irreversible with fl-ecdysone incubation and removal but reversible with juvenile hormone incubation and removal. With ft-ecdysone, the rate of the increase in polymerase I activity paralleled the kinetics of ecdysone binding to discs and increases in the rate of precursor incorporation into RNA. A model to explain the increased activity of RNA polymerase I is presented. A modulation in transcription resulting from exposure of tissues to steroid hormones has been reported in a number of different systems (1-3). The imaginal discs of Drosophila melanogaster are similar in this respect (4, 5) and offer a good opportunity to study the mechanisms of hormonemediated changes in transcription. They can be isolated in gram quantities, and when exposed to physiological concentrations (6) of the molting hormone fl-ecdysone ((be) in vitro, undergo morphological transformations identical to those which occur during metamorphosis (7). The fle-induced morphogenesis is reversibly inhibited by juvenile hormone (JH) (5).The exact biochemical steps leading to disc morphogenesis are obscure. However, observations concerning hormone binding (8) provided by Zoecon Corp. DEAE-Sephadex was washed according to the method of Burgess (12). The buffer (TGMED buffer) used to isolate disc nuclei and to solubilize and chromatograph the polymerases was composed of 50 mM TrisHCl pH 7.9, 5 mM MgCl2, 0.1 mM Na2EDTA, 0.5 mM dithiothreitol, and 25% glycerol (v/v). TGED buffer is TGMED without MgC12.Isolation and Partial Purification of Disc RNA Polymerases. The method used to isolate and partially purify disc RNA polymerases was a modification of those described by Roeder and Rutter (13) and Phillips and Forrest (14). Discs (usually about 600,000), incubated as described below, were washed twice in Ringer's solution at 40 by centrifugation at 500 rpm in a clinical centrifuge. The discs were then resuspended in 5.0 ml of TGMED buffer and Nonidet P-40 was added to a final concentration of 0.5%. The discs were homogenized with five to six gentle strokes using the tightfitting (B) pestle of a Dounce homogenizer, and the homogenate was centrifuged at 16,000 X g for 5 min. The supernatant was decanted and the nuclear pellet frozen on dry ice.The nuclei were stored at -700 for usually less than 1 week. To solubilize the polymerases, 3.0 ml of TGMED were added to the nuclei, and this mixture was shaken until homogeneous. One-tenth volume of 4.0 M (NH4)2SO4 was then added with shaking, and the resulting viscous lysate sonicated at maximum setting for four 10-sec pulses using the Biosonic IV s...

show abstract

New Inhibitors of RNA Synthesis in vitro Isolated from Escherichia coli

Cited by 3 publications

References 16 publications

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

RNA Polymerase of Escherichia coli

Effect of insect hormones on RNA polymerases of mass-isolated imaginal discs of Drosophila melanogaster cultured in vitro.

Contact Info

Product

Resources

About