Abstract
BackgroundThe viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product. Since level and fidelity of transcription primarily determines the overall efficiency, all factors contributing to their decrease should be identified and optimized. Among many observed processes, non-programmed insertion/deletion (indel) of nucleotide during transcription (slippage) occurring at homopolymeric A/T sequences within a gene can considerably impact its expression. To date, no comparative study of the most utilized Escherichia coli and T7 bacteriophage RNA polymerases (RNAP) propensity for this type of erroneous mRNA synthesis has been reported. To address this issue we evaluated the influence of shift-prone A/T sequences by assessing indel-dependent phenotypic changes. RNAP-specific expression profile was examined using two of the most potent promoters, ParaBAD of E. coli and φ10 of phage T7.ResultsHere we report on the first systematic study on requirements for efficient transcriptional slippage by T7 phage and cellular RNAPs considering three parameters: homopolymer length, template type, and frameshift directionality preferences. Using a series of out-of-frame gfp reporter genes fused to a variety of A/T homopolymeric sequences we show that T7 RNAP has an exceptional potential for generating frameshifts and is capable of slipping on as few as three adenine or four thymidine residues in a row, in a flanking sequence-dependent manner. In contrast, bacterial RNAP exhibits a relatively low ability to baypass indel mutations and requires a run of at least 7 tymidine and even more adenine residues. This difference comes from involvement of various intrinsic proofreading properties. Our studies demonstrate distinct preference towards a specific homopolymer in slippage induction. Whereas insertion slippage performed by T7 RNAP (but not deletion) occurs tendentiously on poly(A) rather than on poly(T) runs, strong bias towards poly(T) for the host RNAP is observed.ConclusionsIntrinsic RNAP slippage properties involve trade-offs between accuracy, speed and processivity of transcription. Viral T7 RNAP manifests far greater inclinations to the transcriptional slippage than E. coli RNAP. This possibly plays an important role in driving bacteriophage adaptation and therefore could be considered as beneficial. However, from biotechnological and experimental viewpoint, this might create some problems, and strongly argues for employing bacterial expression systems, stocked with proofreading mechanisms.
Highlights
The viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product
An incredible propensity for slippage on many homopolymeric regions within a given gene found for T7 and E. coli RNA polymerases (RNAP) resulted in a production of a broad spectrum of protein variants with some aminoacid alterations [16]
Productive transcriptional slippage at such sites involves unwinding-rewinding of the RNA:DNA hybrid which is not sensed by RNAP active site and thereby does not lead to the enzyme’s backtracking and mRNA correction
Summary
The viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product. No comparative study of the most utilized Escherichia coli and T7 bacteriophage RNA polymerases (RNAP) propensity for this type of erroneous mRNA synthesis has been reported To address this issue we evaluated the influence of shift-prone A/T sequences by assessing indel-dependent phenotypic changes. Random fluctuation in promoter activity influenced by intrinsic and extrinsic factors causing cell-to-cell variability in the mRNA level during expression of an individual gene is referred to as genetic noise [1,2,3,4]. Studies focused on influence of the A/T homopolymeric regions on helix structures revealed their unusual properties, modifying the normal conformation of B-DNA model [19,20,21,22,23] In this light the poly(A/T) tracts are considered as structures potentially promoting a less accurate transmission of genetic information. A plausible model of the nucleotide misincorporation during T7 phage, yeast, Thermus thermophilus and E. coli RNAP transcription is the template strand misalignment by temporary flipping-out of a base [17, 27,28,29]
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