Control of transcription arrest in intron 1 of the murine adenosine deaminase gene.
Transcription arrest plays a key role in the regulation of the murine adenosine deaminase (ADA) gene, as well as a number of other cellular and viral genes. We have previously characterized the ADA intron 1 arrest site, located 145 nucleotides downstream of the transcription start site, with respect to sequence and elongation factor requirements. Here, we show that the optimal conditions for both intron 1 arrest and overall ADA transcription involve the addition of high concentrations of KCl soon after initiation. As we have further delineated the sequence requirements for intron 1 arrest, we have found that sequences downstream of the arrest site are unnecessary for arrest. Also, a 24-bp fragment containing sequences upstream of the arrest site is sufficient to generate arrest downstream of the adenovirus major late promoter only in the native orientation. Surprisingly, we found that deletion of sequences encompassing the ADA transcription start site substantially reduced intron 1 arrest, with no effect on overall levels of transcription. At the same time, deletion of sequences upstream of the TATA box had no significant effect on either process. We believe the start site mutations have disrupted either the assembly or the composition of the transcription complex such that intron 1 site read-through is now favored. This finding, coupled with the increase in overall transcription after high-concentration KCl treatment, allows us to further refine our model of ADA gene regulation.
Kash
S F
SF
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
Kellems
R E
RE
D000243:Adenosine Deaminase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D011189:Potassium Chloride; D012319:RNA Polymerase II; D000243:Adenosine Deaminase