Gyrase and its associated proteins

Abstract

During transcription, the movement of RNA polymerase generates increasing torsional strain in the template DNA, resulting in positive supercoils ahead of the enzyme and negative supercoils behind it. Efficient progression of the transcription machinery requires the removal of these supercoiling domains by the coordinated action of topoisomerases. DNA gyrase functions ahead of the transcription complex to remove positive supercoils. There may be additional proteins associated with gyrase that affect the topology of the template DNA during transcription. In our laboratory, a direct physical interaction between gyrase and RNA polymerase has been observed in mycobacteria. This interaction may greatly facilitate the efficient removal of transcription driven supercoils from the template. Since the interaction is strong in mycobacteria, studies have been extended to E. coli to understand the functional assembly of the two proteins and the effects of any other proteins that may play a role in the gyrase–RNA polymerase interaction. The first part of the report deals with the effect of a protein named MreB (an actin homolog in bacteria) on gyrase and RNA polymerase. The physical interaction of MreB with DNA gyrase and the effects of this interaction on the supercoiling reaction and on in vitro transcription are described. In the next section, attempts have been made to analyze probable gyrase interacting proteins in Mycobacterium smegmatis. These proteins might play a crucial role in the transcription–topology connection and in gyrase modulation. For this purpose, affinity purification and proteomic approaches have been employed. In the third section of the report, the purification of recombinant GyrA, GyrB, gyrase holoenzyme, and wild type RNA polymerase of Mycobacterium smegmatis is described.