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Nucleic Acids Research, 1991, Vol. 19, No. 8 1783-1790
© 1991

MOLECULAR BIOLOGY

Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2

Orna Resnekov+, Ronald Pruzan§ and Yosef Aloni*

Department of Molecular Genetics and Virology, The Weizmann Institute of Science Rehovot 76100, Israel

* To whom correspondence should ba addressed

Received January 24, 1991. Revised March 27, 1991. Accepted March 27, 1991.

Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell-free system. The results show that RNA polymerase II can halt transcription elongation at a T rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch.

+Present addresses: Department of Bacteriology, Karolinska Institute, Box 60480, 510401 Stockholm, Sweden

§Present addresses: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA


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