T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates

Abstract

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 59 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 59 end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 59 ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 59 end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 59 end heterogeneity in combination with the 39 end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…A GCG sequence was added at the beginning of the transcript to minimize the 5Ј heterogeneity of the RNA population (29). Transcription and RNA purifications steps were performed as described previously (30).…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…The biggest limitation to preparing tRNA by in vitro transcription is that the products frequently possess 3′ and/or 5′ sequence heterogeneities [20,25,26]. These must be eliminated by either tedious purification at reduced yields, or by use of ribozyme sequences to excise the full-length tRNA from flanking regions [27,28].…”

“…For the aminoacyl transfer reaction pathway, the relative standard free energies can be calculated relative to the standard free energy of the E•AA~AMP intermediate using the following equations: (26) (27) The Gibbs activation energy for each reaction can be calculated by subtracting the standard free energy for the enzyme complex preceding the transition state from that of the transition state. All free energies are calculated assuming a standard state of 1 M ATP, 1 M amino acid, and 1 M pyrophosphate.…”

Polymerization of nontemplate bases before transcription initiation at the 3′ ends of templates by an RNA-dependent RNA polymerase: An activity involved in 3′ end repair of viral RNAs

“…Such a mechanism has been proposed to explain the 5Ј end heterogeneity of RNA transcripts produced by the DNA-dependent RNA polymerases of E. coli and T7 bacteriophage (39)(40)(41). However, the templates in those studies contained homooligomeric nucleotides not found on the satC templates truncated by 3 or 4 bases.…”