T7 RNA-POLYMERASE PRODUCES 5' END HETEROGENEITY DURING IN-VITRO TRANSCRIPTION FROM CERTAIN TEMPLATES

The use of T7 RNA polymerase to prepare large quantities of RNA of a p articular sequence has greatly facilitated the study of both the struc ture and function of RNA. Generally, it has been believed that the pro ducts of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5' end sequence and foun d that, for those molecules that begin with multiple, consecutive guan osines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5' end heterogenei ty, two tRNA templates designed to have either four or five consecutiv e guanosines at their 5' ends had more than 30% of their total transcr iptional products extended by at least one untemplated nucleotide at t heir 5' end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5' e nd heterogeneity in combination with the 3' end heterogeneity common t o T7 transcriptions results in a mixture of RNA molecules even after r igorous size purification.