Construction of a novel RNA-transcript-trimming plasmid which can be used both in vitro in place of run-off and (G)-free transcriptions and in vivo as multi-sequences transcription vectors

Abstract

We have constructed a new transcription system that allows trimming of both 5' and 3'-termini of any RNA transcripts by means of cis-acting ribozyme activities. The vector consists of a promoter, '5' Processing Ribozyme', any DNA template to be transcribed, and '3' Processing Ribozyme' sequences. When the vector possessing T7 promoter was tested in vitro, the transcription efficiency from the circular template was over ten-fold higher than using linearized template (run-off transcription). Further, since uniform RNAs with defined 5'- and 3'-ends can be produced, this strategy complements the conventional run-off transcription. Also the 5'-/3'-trimmed uniform RNA can function as a reporter in elucidating transcription factors and promoter regions in vitro, this strategy can replace the widely used (G)-free transcription (Sawadogo and Roeder (1985) Proc. Natl. Acad. Sci. USA 82, 4394-4398). With this strategy, in addition to the advantage that the template DNA need not be linearized prior to transcription, a cytidine-minus sequence is no longer necessary for quantitative analysis of transcription factors. Since any sequences including those of RNA virus can be inserted between the '5' Processing Ribozyme' and the '3' Processing Ribozyme' sequences, and the entire unit can be inserted into any genes under active transcription, this construct is useful like that of Dzianott and Bujarski ((1989) Proc. Natl. Acad. Sci. USA 86, 4823-4827) for RNA virologists because these strategies provide RNA transcripts without heterologous sequences which may greatly diminish infectivity. Moreover, since the construct can also be used in vivo, multi-transcripts such as trans-acting ribozymes targeted to various sites would be produced by concatenating the entire units in tandem.