Partial purification of stable transcription complexes with cloned tRNA genes of XXXX .

@entity3208 @entity969 @entity2975 @entity72404 @entity72405 @entity8660 @entity60097 @entity238
The specific transcription of a cloned @entity238 @entity72404 gene and a @entity72405 gene by extracts from a homologous embryonic cell line showed lag periods of about 30 min before maximum rates were reached. This lag appeared to represent the time to form an active transcription complex. Thus, when extracts were incubated with template DNA for 30 min at 22 degrees C and stored in the cold, the subsequent transcription rate was linear with time and without a lag. After ultracentrifugation of a preincubated reaction mixture on a @entity969 step gradient consisting of 20, 30, 40, and 60% shelves, about 40% of the transcription activity in the extract was found in the 40% shelf. This fraction formed almost exclusively RNA I, the unprocessed tRNA gene transcript, and transcription required only addition of @entity8660 . The rate of formation of RNA by the 40% @entity969 fraction was linear against time, with no lag, and linear with the quantity of fraction. The yield of activity isolated on the gradient was directly proportional to the quantity of cloned gene in the preincubation mixture. At a limiting concentration of the gene in the preincubation mixture, the turnover number of the isolated complex was approximately 50 transcripts/gene/h. @entity3208 - @entity2975 gel electrophoresis of fractions containing the complex still showed many bands, although the complex activity was greatly purified compared to the extract. From the sedimentation behavior of the isolated active transcription complex and from its stability and transcriptional properties, we conclude that the 40% @entity969 fraction contains an active transcription complex containing a cloned tRNA gene, @entity60097 , and the accessory protein factors required for transcription.
