A mutation in T7 RNA polymerase that facilitates promoter clearance

Abstract

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8 -12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.abortive transcription ͉ in vitro RNA synthesis

New Insights into the Mechanism of Initial Transcription

“…Although simple notions of energetic stresses focus on the nucleic acid, for example, DNA scrunching or the inherent instability of short hybrids, mutation in the protein of proline to a leucine at position 266 (P266L), distant from both the hybrid and the promoter binding interface, has been discovered to decrease dramatically the abortive profile of T7 RNA polymerase on otherwise highly abortive sequences (8). How does the single point mutant P266L alter the energetics of initial transcription?…”

“…In the model RNA polymerase from bacteriophage T7, however, a single point mutation (P266L), distant from the hybrid, dramatically reduces the relative production of released abortive transcripts (8), pointing to a more complex process involving the protein. Another popular model for abortive complex instability is the energetic stress of DNA bubble expansion and/or the accommodation of increasing DNA bulk within the protein: scrunching (9 -11).…”

“…The initial report of this mutation indicated that the mutant bound the promoter more weakly than wild type enzyme, suggesting that promoter release was a key element of abortive instability. It was proposed that an earlier release of promoter contacts allowed an earlier transition to a stable elongation complex (8).…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…Milligram quantities of 2F-adenine labeled and unlabeled Gsw apt (73-nt) were synthesized by in vitro transcription from linearized plasmid DNA (TAA TAC GAC TCA CTA TAG GGA CTC ATA TAA CTG CGT GGA TAT GGC ACG CAG GTT TCT ACC GGG CAC CGT AAA TGT CCG ACT ATG GGT CCC) using the P266L mutant of T7 RNA polymerase (Guillerez et al 2005). The in vitro transcription (total volume 18 mL) was performed in 100 mM Trisglutamate buffer pH 8.2, 25 mM Mg(OAc) 2 , 25 mM NTPs (22 % A, 26 % C, 29 % G, 23 % U; for 15 N-labeling, 15 N-GTP and 15 N-UTP were used), 20 mM DTT, 2 mM spermidine, 50 ng/lL DNA (linearized using SmaI), and 70 lg/ mL P266L.…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…The most powerful feature of the method is that tRNAs of virtually any sequence can be prepared, with the caveat that transcription yields are considerably diminished for transcripts initiating with nucleotides other than G. This limitation has recently been overcome through the development of a variant T7 RNA polymerase with reduced specificity for the first nucleotide [22]. Most commonly, the tRNA gene of interest is inserted into the multiple cloning site of an expression plasmid, downstream from the T7 RNA promoter.…”