Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli

M. Zuhaib Qayyum,Debashish Dey,Ranjan Sen

doi:10.1074/jbc.m115.701268

Abstract

NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlappingnut(NusA-binding site) andrut(Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at therutsite that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.

Highlights

The bacterial transcription terminator, Rho, functions as a hexameric RNA-dependent ATPase that is capable of translocating along the RNA
We have provided the following evidence to establish that the transcription elongation factor NusA functions as a general antagonist of Rho by competing for the same or overlapping sites on the mRNA. (i) We have shown that the two NusA-SKK domain (RNA binding domain) mutants as well as the isolated SKK fragment are capable of inhibiting Rho-dependent termination both in vitro and in vivo, and this inhibition was nut site-dependent (Figs. 2 and 3)
SKK domain mutations increase the affinity for the nut site (Fig. 2F). (ii) This inhibition originates from the direct competition between overlapping nut and rut sites (Fig. 4), which delays the Rho loading onto the rut sites (Fig. 5). (iii) The existence of direct competition between Rho and NusA was shown during the antitermination of Rho-dependent termination at the rRNA operon (Figs. 6 and 7) and as a part of the feedback mechanism of the rho expression (Fig. 10). (iv) high resolution tiling microarrays revealed the genome-wide presence of overlapping targets of NusA and Rho, indicating the existence of NusA-mediated antagonism of Rho in many operons as well as in different intergenic regions

Summary

Introduction

The bacterial transcription terminator, Rho, functions as a hexameric RNA-dependent ATPase that is capable of translocating along the RNA. NusA Dissociation Assays in the Presence of Cold Competitors—For measuring the binding strength of WT and mutant derivatives of NusA to the nut site in the presence of a stalled EC, transcription reactions were performed in the presence of 100 nM lac repressor to form a roadblocked EC at the lacO site present downstream of various terminators.

Results

Conclusion