Mechanism, structure and regulatory activity of pyrR regulatory RNA element and its mutants in the presence and absence of uracil.
(A) Schematic representation of the proposed mechanism of regulation of pyr operon by pyrR RNA element. In the presence of UMP, PyrR binds to the pyrR RNA and results in the formation of a premature terminator, disrupting the anti-terminator formed when UMP is low, resulting in transcription termination. (B) RNA-Seq coverage map across the pyr operon (SP_1278–1276) showing premature transcription termination and consequently decreased expression of its genes downstream of the pyrR regulator when grown in defined medium (CDM) in the presence of uracil (yellow) compared to the absence of uracil (blue). Coverage was normalized to the number of uniquely mapped reads in each library. TSSs are in green and the size represents the log transformation of the processed/unprocessed ratio and TTSs are in red and size represents the log transformation of the coverage. (C) qRT-PCR determining the expression of the first genes in the pyr operon (SP_1278) in the presence and absence of uracil validates the RNA-Seq observation. Expression is relative to the qPCR internal control. (D) Secondary structure of the S. pneumoniae pyrR RNA regulatory element in ‘off’ conformation. Boxed in red and yellow are bases that were deleted in M1 and M3 mutations, respectively. Bases boxed in green were replaced by indicated bases to make mutation M2. Highlighted in grey are nucleotides that would base pair to form the anti-terminator when the riboswitch is in the “on” conformation. (E) A representative qRT-PCR quantification of the expression of SP_1278 transcript from pyrR RNA mutant strains cultured in defined medium with or without uracil, corresponding to the regulatory activity of pyrR RNA mutants. While the WTc (wild type with chloramphenicol resistance cassette) decreases expression in the presence of uracil, M1 is insensitive to the ligand. M2 and M3 result in either constitutive or reduced expression of the pyr operon. ~2 fold higher expression in M1 compared to WTc in the absence of uracil could be the result of endogenous uracil having a slight inhibiting effect on the wild type. Expression is relative to the qPCR internal control. Error bars represent standard error of the mean across three technical replicates.