The use of T7 RNA polymerase to prepare large quantities
of RNA of a particular sequence has greatly facilitated
the study of both the structure and function of RNA. Generally,
it has been believed that the products 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 found that, for those molecules that begin
with multiple, consecutive guanosines, the transcriptional
products are far from homogenous. Although a template beginning
with GCG showed no detectable 5′ end heterogeneity,
two tRNA templates designed to have either four or five
consecutive guanosines at their 5′ ends had more
than 30% of their total transcriptional products extended
by at least one untemplated nucleotide at their 5′
end. By simply reducing the number of consecutive guanosines,
the heterogeneity was reduced significantly. The presence
of this 5′ end heterogeneity in combination with
the 3′ end heterogeneity common to T7 transcriptions
results in a mixture of RNA molecules even after rigorous
size purification.