Einfluß der Temperatur auf die Transkription der T3‐DNA durch T3‐spezifische RNA‐Polymerase in zellfreien Extrakten von Escherichia coli CRT266

Abstract

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.

A simple method of distinguishing the bacterial viruses T3 and T7, and a critical reevaluation of their heterologous and homologous exclusion

“…To the slightly viscous suspension distilled glycerol was added to 55 vol% and the mixture was kept at -25 "C. Enzyme activity was stable for at least 1 month. RNA polymerase activity in these crude extracts was determined as previously described (MUSIELSKI et al 1977). The crude extracts contained less than 106 viable bacteria per ml, an amount which does not interfere with the assay of T3-specific RNA polymerase.…”

“…Transcription was started by the addition of 0.1 ml of plasmolysed infected E. coli DG 156 at 30 "C. At the times indicated, 0. I ml aliquots were removed to determine the acid insoluble radioactivity (MUSIELSKI et al 1977).…”