More mistakes by T7 RNA polymerase at the 5′ ends of in vitro-transcribed RNAs

Abstract

In vitro transcription with T7 RNA polymerase is one of the most powerful tools in RNA research. This is mainly linked to the easy preparation of the enzyme, the quite unlimited range of sizes and sequences of the RNA that can be synthesized, as well as the efficiency and accuracy of synthesis. So far only two major limitations are common knowledge, namely poor transcription efficiency of non-G-rich initial sequences (Dunn & Studier, 1983) and 3′-end heterogeneities of the transcripts by 1 or 2 nt (Milligan et al., 1987; Draper et al., 1988; Kholod et al., 1998). These drawbacks have been overcome in most cases by improving the initiating sequence and/or purifying the transcription products to single-nucleotide resolution when necessary. The Uhlenbeck laboratory has published the first evidence for shifty errors by the T7 RNA polymerase at the very 5′ end of the synthesized RNA, an as yet unsuspected event (Pleiss et al., 1998). They demonstrated that in the particular case of tRNA sequences starting with G-rich stretches, the enzyme incorporates additional nontemplated G residues in up to 30% of the transcripts. The authors point to the fact that these errors, in combination with the 3′-end heterogeneity common to T7 transcripts, may be crucial, for instance, for functional studies of tRNAs, because some in vitro-transcribed tRNA molecules of rigorously correct final size may be erroneously considered as functional.