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 promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Here, we compare in vitro and in vivo the yield of long transcripts from T7 promoters, with two different ITSs, the T7 gene10 and the lactose operon ITSs, and two different T7 RNA polymerases, the wild-type and a 2.7-fold slower mutant (G645A). The use of non-cognate ITS and/or slow polymerase decreases the yield of long transcripts in vitro and in vivo in a parallel fashion, with low polymerase speed and non-cognate ITS acting synergistically. In vitro, this decrease is mirrored by an increase in the average number of abortive cycles the enzyme undergoes before leaving the promoter; specifically, with the G645A mutant, transcript release is favored at any ITS position, whereas with the lac ITS it is particularly frequent at positions five and six following the incorporation of uridine residues. Hence, the more abortive cycles per long transcript synthesis in vitro, the lower the yield of long transcripts in vitro or in vivo. We conclude that the duration of abortive cycling can limit long transcript synthesis in vivo, as in vitro. Under conditions where cycling is minimal (wild-type 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.
Lopez
P J
PJ
Laboratoire de Génétique Moléculaire (CNRS URA 1302), Ecole Normale Supérieure, Paris, France.
Guillerez
J
J
Sousa
R
R
Dreyfus
M
M
D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012347:RNA, Transfer, Arg / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000235:genetics
D036022:Capsid Proteins; D012347:RNA, Transfer, Arg; D014764:Viral Proteins; C104967:gene 10 protein, phage T7; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase