Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Abstract

E. coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). Gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.

Mechanism of transcription initiation and promoter escape by E . coli RNA polymerase

“…Instead, we find that the primary difference between promoter OCs with very different lifetimes is in the range of the lengths of the abortive RNAs produced by the fraction of nonproductive ITC at the promoter. It is possible that late-acting E. coli regulatory factors analogous to bacteriophage T7gp2 (55,56) exist that interact selectively with the different downstream structures of OCs with different lifetimes (1,29) to regulate initiation, but none have been discovered. We therefore propose that the longer abortive RNAs produced by long-lived, stable promoter OCs play regulatory roles.…”

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

“…For in vitro footprinting experiments, dsDNA promoter fragments were prepared from 100-nucleotide oligonucleotides corresponding to the À65/+35 promoter positions, as described (Mekler et al 2011). In each case, either the nontemplate or template strand oligonucleotide was labeled with [g-32 P]-ATP at its 59 end.…”

The sabotage of the bacterial transcription machinery by a small bacteriophage protein

“…3 In previous work, we studied the molecular and structural basis of the mechanism by which T7 Gp2 inhibits the bacterial RNAp and our analyses revealed that Gp2 uses a multipronged strategy to inactivate the bacterial RNAp. 4,5 Such studies have broad implications not only in elucidating novel paradigms of bacterial transcription regulation, but also for uncovering strategies for development of novel antibacterial compounds targeting bacterial RNAp.…”