Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B

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Abstract
Nuclei which were isolated from SV40 infected cells with a hypotonic detergent-free buffer were used to establish in vitro conditions which lead to transcription-termination at the attenuation site of SV40. This system allowed us to identify regulatory elements involved in transcription-termination by RNA polymerase B transcribing SV40. Transcription-termination at the attenuation site was found to be ionic strength dependent. Efficient termination occurred at low (100 mM NaCl) but not at high (100 mM (NH 4 ) 2 SO 4 or 300 mM NaCl) ionic strength. When nuclei were prewashed with 300 mM NaCl, the efficiency of transcription-termination was low even when transcription was carried out at low ionic strength (100 mM NaCl). Efficient transcription-termination in the high salt prewashed nuclei was reconstituted by complementation with a high salt (300 mM NaCl) soluble factor extracted from nuclei of uninfected cells. In addition, the efficiency of transcription-termination was significantly reduced when ITP replaced GTP in the transcription reaction mixture. Our data indicate that a nuclear factor and RNA secondary structure are essential regulatory elements involved in transcription-termination by RNA polymerase B.