Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, sigma-subunit and GreB.
Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs. The process of transcription is accompanied by complex structural rearrangements of RNAP. Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP. Using in vitro selection methods, we obtained 13 classes of ssDNA aptamers against Escherichia coli core RNAP. The minimal nucleic acid scaffold (an oligonucleotide construct imitating DNA and RNA in elongation complex), rifampicin and the sigma70-subunit inhibited binding of the aptamers to RNAP core but did not affect the dissociation rate of preformed RNAP-aptamer complexes. We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel. In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP-aptamer complexes. This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP-aptamer complexes by inducing conformational changes in the channel. We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investigating the conformational flexibility of the enzyme.
Kulbachinskiy
Andrey
A
Public Health Research Institute, Newark, New Jersey, USA. akulb@img.ras.ru
Feklistov
Andrey
A
Krasheninnikov
Igor
I
Goldfarb
Alex
A
Nikiforov
Vadim
V
D001483:Base Sequence; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012293:Rifampin / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D035602:Transcriptional Elongation Factors
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin