Novel antitermination mechanisms of transcription

Abstract

Bacteriophage T7 infection of $\it{Escherichia coli}$ is uniquely slow, taking about 10 minutes, as linear phage DNA is injected into host by DNA pulling action of transcribing RNA polymerase. A strong intrinsic terminator $T\Phi$ encoding an RNA hairpin-oligo(U), however, is located at a three-fifth position of T7 genome and halts the phage DNA injection. This terminator should be inactivated for resumption of injection and expression of $T\Phi$-downstream promoter-less genes $\it{11}$ and $\it{12}$, but its regulatory mechanism has not been known to date. Two novel mechanisms are provided in this study for antitermination of $T\Phi$, both involving modulation of RNA secondary structure. Firstly, the oligo(U) sequence exerts an antitermination activity on terminator $T\Phi$ via allowing elongation complexes to pause. The pausing is observed three base-pairs upstream of termination site in a single-round transcription reaction. This pausing is caused by the oligo(U) sequence rather than the RNA hairpin, and RNA polymerase is backtracked with three 3`-end RNA residues protruded from the active site due to weak interactions between rU and dA. The backtracked pausing favors formation of a thermodynamically more stable antiterminator structure rather than an incomplete terminator RNA structure, resulting in inactivation of terminator $T\Phi$. In the antiterminator RNA, the 5` half of the terminator RNA was sequestered by a further upstream sequence in a $\it{cis}$-acting manner. Secondly, we show that abortive initiation affects termination in transcription of bacteriophage T7 gene $\it{10}$. Specifically, abortive transcripts produced from promoter $\Phi$10 exert $\it{trans}$-acting antitermination activity on terminator $T\Phi$both $\it{in vitro}$ and $\it{in vivo}$. Following abortive initiation cycling of T7 RNA polymerase at $\Phi$10, short G-rich and oligo(G) RNAs are produced and both specifically sequester 5- and 6-nucleotide C+U stretch sequences, cons...