THE LOW PROCESSIVITY OF T7 RNA-POLYMERASE OVER THE INITIALLY TRANSCRIBED SEQUENCE CAN LIMIT PRODUCTIVE INITIATION IN-VIVO

In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the p romoter. With the bacteriophage T7 enzyme, the extent of abortive tran scription varies with the nature of the ITS and with the elongation sp eed of the polymerase. Here, we compare in vitro and in vivo the yield of long transcripts from T7 promoters, with two different ITSs, the T 7 gene10 and the lactose operon ITSs, and two different T7 RNA polymer ases, the wild-type and a 2.7-fold slower mutant (G645A). The use of n on-cognate ITS and/or slow polymerase decreases the yield of long tran scripts in vitro and in vivo in a parallel fashion, with low polymeras e speed and non-cognate ITS acting synergistically. In vitro, this dec rease is mirrored by an increase in the average number of abortive cyc les the enzyme undergoes before leaving the promoter; specifically, wi th the G645A mutant, transcript release is favored at any ITS position , whereas with the lac ITS it is particularly frequent at positions fi ve and six following the incorporation of uridine residues. Hence, the more abortive cycles per long transcript synthesis in vitro, the lowe r the yield of long transcripts in vitro or in vivo. We conclude that the duration of abortive cycling can limit long transcript synthesis i n vivo, as in vitro. Under conditions where cycling is minimal (wild-t ype polymerase, gene10 ITS), T7 promoter drives the synthesis of three long transcripts per second at 37 degrees C in vivo, a figure higher than for any Escherichia coli promoter. (C) 1997 Academic Press Limite d.