Relaxed Rotational and Scrunching Changes in P266L Mutant of T7 RNA Polymerase Reduce Short Abortive RNAs while Delaying Transition into Elongation

Abstract

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4–7 nt) while marginally increasing long abortives (9–11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K d by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1–2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis

“…DNAs used for the assembly of transcription complexes were purchased as single-stranded oligonucleotides (Eurofines). RNA constructs were synthesized by in vitro transcription by T7 P266L RNA polymerase (28), using PCR products as templates, and purified as described (19).…”

Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis

“…DNAs used for the assembly of transcription complexes were purchased as single-stranded oligonucleotides (Eurofines). RNA constructs were synthesized by in vitro transcription by T7 P266L RNAP (Tang et al, 2014), using PCR-amplified linear DNAs as templates, and purified as described Said et al (2017).…”

Native Purification and Analysis of Long RNAs

“…We use the T7 RNA polymerase mutant P266L, which is known to produce short abortive transcripts less frequently than wild-type T7 polymerase (Tang et al, 2014). If using a commercial T7 RNA polymerase, we suggest following the manufacturer instructions.…”

“…The use of tags, generally added to the 3′ end of target RNAs, ensures capturing a homogeneous population of full-length molecules. In our method, we achieve a similar goal by using a T7 polymerase construct that rarely produces short abortive transcripts (Tang et al, 2014), and centrifugal filtration and size-exclusion chromatography (SEC) as final polishing steps in purification. Not using tags may simplify cloning design and avoid inclusion of nonnative sequences that may interfere with structure formation of the target RNA.…”