Cell free extracts were prepared from E . coli CRT266 9 min after infection with T3 phages. RNA synthesis in these extracts is almost entirely due to T3 RNA polymerase. The inactivation of T3 RNA polymerase in these extracts proceeds rapidly at 42 "C. 90% of the activity is lost within 10 min a t this temperature. Under conditions where the formation of a stable initiation complex with T3 DNA is possible, i.e., in the presence of GTP, ATP, and UTP the T3 RNA polymerase becomes protected against heat inactivation losing only 10% of its activity during a n exposure to 42 "C for 10 min. Studies on the time course of RNA synthesis have shown that reinitiation is still possible at 37 "C and 42 "C. At 44 "C, however, RNA synthesis stops abruptedly after 3 min indicating that reinitiation does no longer take place. The elongation of already initiated T3 RNA chains is rather resistant to heat. At 44 "C the same elongation rates are observed as a t 37 "C and 42 "C, respectively.

Ihnethylsulfoxide (DMSO) (this paper) as well as KCI inhibit both the transcription by Thpecific RNA polymerase (CHAERABORTY et al. 1973, MCALLISTER et al. 1973, MUSIELSKI et al. 1970. The mechanism by which these substances act upon the DNA is quite different. While KC1 stabilizes the DNA helix (DOVE and DAVIDSON 1902) DMSO labilizes the latter by decreasing the hydrophobic interactions within the polynucleotide chains (HERSKOVITS 1962, LOBER and SCHUTZ 1972, ZIMMER and LUCK 1972. One should, therefore, expect that DMSO would remove the inhibitory action of KC1 on the transcription by T3-jnduced RNA polymerase and vice versa. That this is indeed the case was shown in a previous report (MUSIELSKI et al. 1976). Siiiiilar observations have also been reported for the transcription of il gal DNA (NAKANISHI et al. 1974). I n order to gain a better understanding of this interrelation the transcription of T3 DEA by T3-induced RNA polymerase was studied in the presence of various c*oncentrations of KCl and DMSO and the results obtained are reported below.T3-induced RNA polymerase was isolated essentially according to CHAKRABORTY ef al. (1973). RNA synthesis was assayed as described previously (MUMELSKI et al. 1977). DMSO was gas chromatographically pure. Experimental details are given in the legend to Fig. 1. DMSO a t concfientrations above 8y0 (v/v) and KC1 exceeding 25 n i~ inhibit the transcription by T3-induced RNA polynierase if added individually. However, if RNA synthesis proceeds in a reaction rnixture containing both DMSO and KCl the action of these substances, i. e., whether inhibitory or stimulatory, depends on the salt to solvent ratio. At low concentrations of DMSO (4 and 8% v/v) no inhibitory effect of the solvent could be noted. Nevertheless, on the addition of salt (25 m n i ) a significant stimulation of the RNA syntheses was observed (Fig. 1). This indicates that under the mentioned conditions a inore favourable conformation of the T3 DNA should have been attained. At higher concentrations of DMSO, i. e., above 8 ) 1 (v/v), when the solvent depresses transcription by T3-induced RNA polymerase, addition of salt still increases KKA syntheses. Though increasing the ionic strength produces some stimulatory effects at all DMSO concentrations tested the curves describing this phenomenon differed. At low DMSO levels the optimal salt concentrations were well defined and exceeding theses concentrations the usual salt inhibition was observed. With increasing amounts of solvent in the assay niixture the optimal ionic strength shifts towards higher values and the optima become less pronounced. With the DMSO concentration above 20% (v/v) practically a plateau is reached at 75 I n M KC1 which decreases only slightly after the salt concentration has surpassed 200 1 x 1~.

Cell free extracts were prepared from E. coli CRT266 9 min after infection with T3 phages. RNA synthesis in these extracts is almost entirely due to T3 RNA polymerase. The inactivation of T3 RNA polymerase in these extracts proceeds rapidly at 42 degrees C. 90% of the activity is lost within 10 min at this temperature. Under conditions where the formation of a stable initiation complex with T3 DNA is possible, i.e., in the presence of GPT, APT, and UTP the T3 RNA polymerase becomes protected against heat inactivation losing only )0% of its activity during an exposure to 42 degrees C for 10 min. Studies on the time course of RNA synthesis have shown that reinitiation is still possible at 37 degrees C and 42 degrees C. At 44 degrees C, however, RNA synthesis stops abruptly after 3 min indicating that reinitiation does no longer take place. The elongation of already initiated T3 RNA chains is rather resistant to heat. At 44 degrees C the same elongation rates are observed as at 37 degrees C and 42 degrees C, respectively.