Oshiro, R. T. | bioRxiv Science

bioRxiv2020

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates global gene expression with NusA

NusA and NusG are transcription elongation factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor, the role of NusG in this process had not been explored. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, {Delta}nusG, and NusA depletion {Delta}nusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1,400 identified intrinsic terminators. The loss of both proteins leads to global misregulation of gene expression. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Moreover, the loss of NusG results in flagella and swimming motility defects.

microbiology↗

bioRxiv2020

Contact with the CsrA core is required for allosteric inhibition by FliW in Bacillus subtilis

The RNA-binding protein CsrA is a post-transcriptional regulator that is encoded in genomes throughout the bacterial phylogeny. In the gamma-proteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, that non-competitively inhibits CsrA activity but the precise mechanism of antagonism is unclear. Here we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition that fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues that surround the critical regulatory CsrA residue N55 and abolished CsrA interaction. Two loss-of-function alleles however mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of these two alleles reversed charge at what appeared to be a salt bridge with the CsrA core region, charge reversal of the CsrA partner residue phenocopied the FliW allele, and charge reversal of both residues simultaneously restored antagonism. We propose a model in which initial interaction between FliW and CsrA is necessary but not sufficient for antagonism which also requires salt bridge formation with, and deformation of, the CsrA core domain to allosterically abolish RNA binding activity. SummaryCsrA is a small dimeric protein that binds RNA and is one of the few known examples of transcript-specific translational regulators in bacteria. A protein called FliW binds to and antagonizes CsrA; despite having a high-resolution three-dimensional structure of the FliW-CsrA complex, the mechanism of non-competitive inhibition remains unresolved. Here we identify FliW residues required for antagonism and we find that the residues make a linear connection in the complex from initial binding interaction with CsrA to a critical salt bridge near the core of the CsrA dimer. We propose that the salt bridge represents an allosteric contact that distorts the CsrA core to prevent RNA binding.

microbiology↗