Sequences required for transcription termination at the intrinsic tI terminator

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Abstract:

The tI terminator is located approximately 280 bp beyond the int gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for tI transcription termination a set of deletion mutants were generated, either from the 5′ or the 3′ end onto the tI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3′ end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at tI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3′ end confirmed the importance of the hairpin stem in protection against exonuclease.

Le terminateur tlest localisé approximativement 280 pb au-delà du gène int, et il possède une structure typique d’un terminateur intrinsèque. Afin d’identifier les séquences requises à la terminaison de la transcription de tl, une série de mutants de délétion ont été générés, aux extrémités 5′ ou 3′, dans la région du gène int. L’efficacité de terminaison a été déterminée en mesurant les niveaux de galactokinase (galK) par buvardage Northern et par terminaison de la transcription in vitro. L’importance de la présence de résidus uridine et de la stabilité de la structure en tige dans la terminaison ont été démontrées. L’ADN non transcrit situé au-delà de l’extrémité 3′ affecte aussi la terminaison. De plus, les séquences présentes en amont ont un léger effet sur la terminaison de la transcription. Les sites de terminaison de l’ARN in vivo sur tlont été déterminés par cartographie S1 et ont été localisés à 8 positions différentes. La maturation moléculaire des transcrits de l’extrémité 3′ a confirmé l’importance de la tige en épingle à cheveux dans la protection contre les exonucléases.

Document Type: Research Article

Publication date: February 2, 2010

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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