“…Desired products from in vitro transcription of CQ1 (an A1G mutant of E. coli tRNA Gln ), CQ10 (a GC 4-69 AU mutant of CQ1), YF0 (yeast tRNA Phe ), CA0 (E. coli tRNA Ala ), and CA2 (a GC 4-69 CG mutant of CA0)+ method used for detecting these aberrant RNA products+ Transcripts prepared with 10% ATPaS and 90% ATP did not change the relative amount or position of the additional bands, indicating that the presence of the phosphorothioate containing NTP did not intrinsically produce the extra bands+ Transcriptions with either CTPaS or UTPaS instead of ATPaS also gave very similar patterns for the extra products, suggesting that the substituted ATPaS did not cause the extra products+ Finally, RNAs transcribed with only the four normal NTPs and [g-32 P] GTP were purified and subjected to partial digestion with RNase T1 to give specific cleavage after guanosines+ The minor bands observed by this method were identical in amount and distribution to those seen using phosphorothioate cleavage+ Two different mechanisms may explain the 59-terminal heterogeneity observed in the transcription products from T7 promoter sequences containing consecutive guanosine residues+ One possibility is that slippage occurs during the initiation processes such that, after the second nucleotide is added to form pppGpG, the dinucleotide can slip back by one residue in the enzyme active site prior to further elongation+ Such a mechanism has been proposed to explain 59-terminal heterogeneity produced by Escherichia coli RNA polymerase when initiating transcription with consecutive adenosines or uridines (Guo & Roberts, 1990;Xiong & Reznikoff, 1993)+ A related possibility is based on the observation that the large amount of abortive initiation products produced by T7 RNA polymerase can serve as primers for future initiation events (Moroney & Piccirilli, 1991)+ If one assumes that abortive products can reinitiate in an incorrect frame, 59 end heterogeneity would result+ More work is needed to clearly define the exact mechanism or mechanisms by which 59-terminal heterogeneity is produced by this polymerase+ Regardless of the mechanism, the presence of 59-terminal heterogeneity greatly complicates the isolation of RNA molecules with both length and sequence homogeneity because of the inevitable 39-terminal heterogeneity that results from premature termination and nontemplated additions of nucleotides by T7 RNA polymerase+ To illustrate this point, one of these tRNAs was subjected to a functional assay+ Using [a-32 P] CTP, CA0 was transcribed to a low specific activity, allowing the transcription products to be purified to single-nucleotide resolution on a polyacrylamide gel (Fig+ 3A)+ Each of the three major transcription products was assayed subsequently for its ability to be aminoacylated by E. coli alanyl-tRNA synthetase+ Because aminoacyl-tRNA synthetases require a 39-terminal CCA sequence in order to covalently attach an amino acid, the fraction of each band with a properly terminated 39 end can be estimated by measuring the amount of tritiated amino acid incorporated into the RNA+ As shown in Figure 3B, approximately 80% of the molecules in the proper sized Line integration of the radioactivity present near one expected adenosine for each RNA+ The major band is present at the left end of the trace, with positions ϩ1, ϩ2, and ϩ3 appearing to the right+ The percentage of radioactivity present in each peak is indicated+ transcript (76-nt long) are able to be aminoacylated+ The transcript one nucleotide shorter was only aminoacylatable to a very low level, whereas the transcript one nucleotide longer reached a level of approximately 55%+ These results correlate well with the RNA sequencing data+ Because none of the detectable 59 end heterogeneity se...…”