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Fig. 6. In vitro transcription of linearized plasmids carrying a variety of ITS, by the WT or P266L T7 RNAP. (A) Sequence of the ITS used; also shown is the length of the runoff transcript produced in each case. The four ITS (Tyr, TyrG, Gly, and Val) are derived from yeast (Tyr, TyrG, Val) or human mitochondria (Gly) tRNA sequences; the corresponding plasmids [a gift from J. Rudinger, Institut de Biologie Moléculaire et Cellulaire (Strasbourg, France); cf. ref. 1] were linearized with BstNI to produce the indicated runoff transcripts. (B) In vitro transcription pattern from the ITS shown in A. Transcription was run as in Fig. 2B, except that [a -³²P]UTP was used as the label instead of [a -³²P]GTP. The ITS and the RNAP used in each case are indicated below and above the corresponding lane, respectively. The lengths of abortive species are indicated beside the corresponding lanes. Bands noted * seem to correspond to minor initiations at positions +2 (CU for Gly and GU for Val; note the comigration with the same species from Tyr and TyrG, respectively). A minor species observed with the Val ITS (**) slowly accumulates during time-course experiments; it corresponds presumably to runoff products that are further extended by T7 RNAP (2–4). Note that in all cases, the use of the P266L mutation results in a marked fading of the abortive transcripts. This reduction stimulates runoff transcription in case where the WT enzyme aborts frequently at positions 5–7 (Tyr, TyrG, and Val; compare also lac). (C) Processivity of the WT enzyme (open circles) and of the P266L mutant (filled circles) over the different ITS shown in A. Processivity values (see text for definition) have been calculated from the intensities of the different bands seen in B.

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