Mechanisms for Defining Supercoiling Set Point of DNA Gyrase Orthologs: I. A NONCONSERVED ACIDIC C-TERMINAL TAIL MODULATES ESCHERICHIA COLI GYRASE ACTIVITY

Elsa M Tretter,James M Berger

doi:10.1074/jbc.m112.345678

Elsa M Tretter, James M Berger

Open Access

https://doi.org/10.1074/jbc.m112.345678

Copy DOI

Abstract

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.

Highlights

The mechanisms controlling DNA supercoiling efficiency by gyrase are not understood
All type IIA topoisomerases employ a similar set of catalytic domains for function; the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit
We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function

Summary

Background

The mechanisms controlling DNA supercoiling efficiency by gyrase are not understood. Results: A nonconserved C-terminal tail in GyrA controls DNA binding, wrapping, and supercoiling set point. In comparing the supercoiling properties of Mycobacterium tuberculosis (Mtb) and E. coli gyrase, we discovered that the isolated GyrA proteins of the two species differ dramatically in their respective abilities to wrap DNA (see accompanying article [46]) Further analysis of this distinction led us to probe the function of the nonconserved stretch of amino acids that follows the CTD in E. coli GyrA (see Fig. 1B). Alterations to the CTD tail have no effect on either basal or DNA-stimulated ATPase activity, but greatly reduce both the rate of negative supercoiling and the final level of superhelical density that can be introduced by gyrase These findings indicate that species-specific appendages to the GyrA CTD can regulate its function in the context of the gyrase holoenzyme, thereby ensuring that ATP turnover is tightly coupled to supercoiling efficiency

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION