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

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 39-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 59 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 39-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+ Here we bring additional evidence for novel types of sly errors by the T7 RNA polymerase at the 59 end of transcripts+ These errors occur (1) at very high frequency (80 to 100% for a given template), (2) on initial sequences other than homopolymeric G stretches, and (3) either by incorporation of a single nontemplated nucleotide (predominantly an A) or omission of the first nucleotide+ Here also, a combination of these errors of the variety of those possible at the 39-end leads to incorrect RNAs, despite their rigorous size purification+