RNA Polymerase Alters the Mobility of an A-Residue Crucial to Polymerase-Induced Melting of Promoter DNA

Abstract

Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position -11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the -11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the -11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position -11 has no major effect on the accessibility of DNA to DNase I or KMnO(4) in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at -11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.

Defining the Molecular Forces That Determine the Impact of Neomycin on Bacterial Protein Synthesis: Importance of the 2′-Amino Functionality

“…The anisotropy decays are best described as the sum of two exponential terms (equation 3). The r(0) values for the biexponential fits range from 0.266 to 0.356, in good agreement with previously reported r(0) values for 2AP in both DNA and RNA, which ranged from 0.266 to 0.377 (22,25,32,40).…”

The essential activities of the bacterial sigma factor

“…In the A -11 binding pocket, a tyrosine (Y253, Fig. 4b) stacks against the purine base (Feklistov and Darst 2011), confirming conclusions from earlier biochemical studies using a base analog at position -11 (Lim et al 2001;Tsujikawa et al 2002a). Following the work of Feklistov and Darst (2011), a structure solved by Ebright and co-workers consisting of nearly full-length T. aquaticus A and a more extensive opencomplex construct confirmed these base-flipping events and indicated 2 additional flipped out bases at positions -6 and +2 (Zhang et al 2012).…”

Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site

“…10 The shorter rotational correlation time (φ 2 ) reflects the internal motion of the 2AP base in the RNA, with the subnanosecond value of φ 2 (0.47 ± 0.09 ns) obtained in the absence of bound drug being characteristic of previously reported values for 2AP residues in short DNA and RNA duplex constructs at temperatures ≥ 20 °C. 4,5,8,10 Note that drug binding increases φ 2 , an observation indicative of a drug-induced decrease in the mobility of the 2AP base at position 1493. We have previously observed a similar trend with regard to the mobility of the base at position 1492.…”

“…The r(0) values for the biexponential fits range from 0.290 to 0.375, in good agreement with previously reported r(0) values for 2AP in both DNA and RNA, which range from 0.266 to 0.377. 4,5,8,10 The longer of the two rotational correlation times (φ 1 ) reflects the overall tumbling of the RNA duplex. 10 The shorter rotational correlation time (φ 2 ) reflects the internal motion of the 2AP base in the RNA, with the subnanosecond value of φ 2 (0.47 ± 0.09 ns) obtained in the absence of bound drug being characteristic of previously reported values for 2AP residues in short DNA and RNA duplex constructs at temperatures ≥ 20 °C.…”

“…By so doing, one is then in a position to use fluorescence-based approaches to characterize the structure, energetics, and dynamics of nucleic acids and their complexes with other biologically relevant molecules. [3][4][5][6][7][8][9][10] Here, we site specifically introduce 2AP into either of two different positions (1492 or 1493) of an RNA oligonucleotide (Ec) that models the E. coli 16 S rRNA A-site (see Figure 1). We determine the impact of each 2AP substitution on the structure and stability of the RNA, and use the intrinsic fluorescence properties of 2AP to characterize the energetics and dynamics associated with the interactions between the rRNA A-site model oligomer and the aminoglycoside antibiotics, paromomycin, geneticin (G418), neomycin, and ribostamycin (see drug structures in Figure 2).…”