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Three inhibitor proteins (I,, I, and 13) were isolated from Escherichia coli. They depress the transcription of E. coli and T3 phage DNA by DNA-dependent RNA polymerase from E. coli and show the following characteristics.1. The inhibitors I, and I3 consist of subunits; I2 behaves as a single protein in dodecylsulfatepolyacrylamide gel electrophoresis.2. Neither the initiation of the transcription of E. coli DNA nor the size of the RNA synthesized is influenced by the inhibitors.3. In an E. coli DNA-dependent system reinitiation of RNA synthesis at high salt concentration seems to be blocked by the inhibitors.4. When inactivated, the inhibitors stimulate RNA synthesis and behave like termination factors giving rise to the production of RNA molecules of discrete size, shorter than those of control experiments.5. It is suggested by hybridization competition experiments that the inhibitors I, and I3 preferentially inhibit ribosomal RNA synthesis.6. The inhibitors also depress the transcription of T3 phage DNA by E. coli RNA polymerase resulting in the synthesis of discrete RNA species smaller than those of control experiments without KCl as well as with 0.16 M KCl.In several laboratories the presence of some protein factors restricting DNA-dependent RNA synthkis have been demonstrated in Escherichia coli. Rho factor isolated by Roberts [l] promotes the release of RNA molecules of discrete size from template DNA. Recently, evidence has been put forward that rho factor and also kappa factor isolated by Schaffer et al. [2] block RNA synthesis rather than act as termination factors [ 3 ] .From E. coli we isolated three inhibitors which can depress RNA synthesis. Though on T3 phage DNA these inhibitors terminate RNA synthesis at some special sites leading to the synthesis of RNA molecules of discrete size, in the E. coli DNA-dependent system they seem to be factors inhibiting RNA synthesis ; they arrest transcription without changing the product.Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6). MATERIALS AND METHODS Crude Purification of InhibitorsPreparation of a Crude Extract. 100 g of E. coli cell paste (E. coli Q 13 grown in 1.3% trypton, 0.7% NaCl with forced aeration until stationary phase at 37 "C) was suspended in 130 ml of buffer A (50 mM Tris . HC1, pH 7.8, 100 mM KC1, 10 mM MgC12) and homogenized by shaking with glass beads at high frequency. After removal of the glass beads by filtration of the homogenate through a sintered glass funnel, cell debris was spun down by low-speed centrifugation (40000 x g , 10 min). In order to remove ribosomes the supernatant was subjected to high-speed centrifugation at 50000 rev./min for 90 min using a Spinco 60 Ti rotor. The supernatant plus a thin DNA film on top of the ribosomal pellet was collected and used for the preparation of the inhibitors.Eur. J. Biochem. 49 (1974)